Apo-2 receptor

ABSTRACT

Novel polypeptides, designated Apo-2, which are capable of modulating apoptosis are provided. Compositions including Apo-2 chimeras, nucleic acid encoding Apo-2, and antibodies to Apo-2 are also provided.

RELATED APPLICATIONS

[0001] This application is a continuation application of Ser. No.10/052,798 filed Nov. 2, 2001, which is a divisional application of Ser.No. 09/079,029 filed May 14, 1998, now issued as U.S. Pat. No.6,342,369, claiming priority under Section 119(e) to provisionalapplication No. 60/046,615 filed May 15, 1997 and provisionalapplication No. 60/074,119 filed Feb. 9, 1998, the contents of all ofwhich are hereby incorporated by reference.

FIELD OF THE INVENTION

[0002] The present invention relates generally to the identification,isolation, and recombinant production of novel polypeptides, designatedherein as Apo-2, and to anti-Apo-2 antibodies.

BACKGROUND OF THE INVENTION

[0003] Apoptosis or “Programmed Cell Death”

[0004] Control of cell numbers in mammals is believed to be determined,in part, by a balance between cell proliferation and cell death. Oneform of cell death, sometimes referred to as necrotic cell death, istypically characterized as a pathologic form of cell death resultingfrom some trauma or cellular injury. In contrast, there is another,“physiologic” form of cell death which usually proceeds in an orderly orcontrolled manner. This orderly or controlled form of cell death isoften referred to as “apoptosis” [see, e.g., Barr et al.,Bio/Technology, 12:487-493 (1994); Steller et al., Science,267:1445-1449 (1995)]. Apoptotic cell death naturally occurs in manyphysiological processes, including embryonic development and clonalselection in the immune system [Itoh et al., Cell, 66:233-243 (1991)].Decreased levels of apoptotic cell death have been associated with avariety of pathological conditions, including cancer, lupus, and herpesvirus infection [Thompson, Science, 267:1456-1462 (1995)]. Increasedlevels of apoptotic cell death may be associated with a variety of otherpathological conditions, including AIDS, Alzheimer's disease,Parkinson's disease, amyotrophic lateral sclerosis, multiple sclerosis,retinitis pigmentosa, cerebellar degeneration, aplastic anemia,myocardial infarction, stroke, reperfusion injury, and toxin-inducedliver disease [see, Thompson, supra].

[0005] Apoptotic cell death is typically accompanied by one or morecharacteristic morphological and biochemical changes in cells, such ascondensation of cytoplasm, loss of plasma membrane microvilli,segmentation of the nucleus, degradation of chromosomal DNA or loss ofmitochondrial function. A variety of extrinsic and intrinsic signals arebelieved to trigger or induce such morphological and biochemicalcellular changes [Raff, Nature, 356:397-400 (1992); Steller, supra;Sachs et al., Blood, 82:15 (1993)]. For instance, they can be triggeredby hormonal stimuli, such as glucocorticoid hormones for immaturethymocytes, as well as withdrawal of certain growth factors[Watanabe-Fukunaga et al., Nature, 356:314-317 (1992)]. Also, someidentified oncogenes such as myc, rel, and E1A, and tumor suppressors,like p53, have been reported to have a role in inducing apoptosis.Certain chemotherapy drugs and some forms of radiation have likewisebeen observed to have apoptosis-inducing activity [Thompson, supra].

[0006] TNF Family of Cytokines

[0007] Various molecules, such as tumor necrosis factor-α (“TNF-α”),tumor necrosis factor-β (“TNF-β” or “lymphotoxin”), CD30 ligand, CD27ligand, CD40 ligand, OX-40 ligand, 4-1BB ligand, Apo-1 ligand (alsoreferred to as Fas ligand or CD95 ligand), and Apo-2 ligand (alsoreferred to as TRAIL) have been identified as members of the tumornecrosis factor (“TNF”) family of cytokines [See, e.g., Gruss and Dower,Blood, 85:3378-3404 (1995); Wiley et al., Immunity, 3:673-682 (1995);Pitti et al., J. Biol. Chem., 271:12687-12690 (1996); WO 97/01633published Jan. 16, 1997]. Among these molecules, TNF-α, TNF-β, CD30ligand, 4-1BB ligand, Apo-1 ligand, and Apo-2 ligand (TRAIL) have beenreported to be involved in apoptotic cell death. Both TNF-α and TNF-βhave been reported to induce apoptotic death in susceptible tumor cells[Schmid et al., Proc. Natl. Acad. Sci., 83:1881 (1986); Dealtry et al.,Eur. J. Immunol., 17:689 (1987)]. Zheng et al. have reported that TNF-αis involved in post-stimulation apoptosis of CD8-positive T cells [Zhenget al., Nature, 377:348-351 (1995)]. Other investigators have reportedthat CD30 ligand may be involved in deletion of self-reactive T cells inthe thymus [Amakawa et al., Cold Spring Harbor Laboratory Symposium onProgrammed Cell Death, Abstr. No. 10, (1995)].

[0008] Mutations in the mouse Fas/Apo-1 receptor or ligand genes (calledlpr and gld, respectively) have been associated with some autoimmunedisorders, indicating that Apo-1 ligand may play a role in regulatingthe clonal deletion of self-reactive lymphocytes in the periphery[Krammer et al., Curr. Op. Immunol., 6:279-289 (1994); Nagata et al.,Science, 267:1449-1456 (1995)]. Apo-1 ligand is also reported to inducepost-stimulation apoptosis in CD4-positive T lymphocytes and in Blymphocytes, and may be involved in the elimination of activatedlymphocytes when their function is no longer needed [Krammer et al.,supra; Nagata et al., supra]. Agonist mouse monoclonal antibodiesspecifically binding to the Apo-1 receptor have been reported to exhibitcell killing activity that is comparable to or similar to that of TNF-α[Yonehara et al., J. Exp. Med., 169:1747-1756 (1989)].

[0009] TNF Family of Receptors

[0010] Induction of various cellular responses-mediated by such TNFfamily cytokines is believed to be initiated by their binding tospecific cell receptors. Two distinct TNF receptors of approximately55-kDa (TNFR1) and 75-kDa (TNFR2) have been identified [Hohman et al.,J. Biol. Chem., 264:14927-14934 (1989); Brockhaus et al., Proc. Natl.Acad. Sci., 87:3127-3131 (1990); EP 417,563, published Mar. 20, 1991]and human and mouse cDNAs corresponding to both receptor types have beenisolated and characterized [Loetscher et al., Cell, 61:351 (1990);Schall et al., Cell, 61:361 (1990); Smith et al., Science, 248:1019-1023(1990); Lewis et al., Proc. Natl. Acad. Sci., 88:2830-2834 (1991);Goodwin et al., Mol. Cell. Biol., 11:3020-3026 (1991)]. Extensivepolymorphisms have been associated with both TNF receptor genes [see,e.g., Takao et al., Immunogenetics, 37:199-203 (1993)]. Both TNFRs sharethe typical structure of cell surface receptors including extracellular,transmembrane and intracellular regions. The extracellular portions ofboth receptors are found naturally also as soluble TNF-binding proteins[Nophar, Y. et al., EMBO J., 9:3269 (1990); and Kohno, T. et al., Proc.Natl. Acad. Sci. U.S.A., 87:8331 (1990)]. The cloning of recombinantsoluble TNF receptors was reported by Hale et al. [J. Cell. Biochem.Supplement 15F, 1991, p. 113 (P424)].

[0011] The extracellular portion of type 1 and type 2 TNFRs (TNFR1 andTNFR2) contains a repetitive amino acid sequence pattern of fourcysteine-rich domains (CRDs) designated 1 through 4, starting from theNH₂-terminus. Each CRD is about 40 amino acids long and contains 4 to 6cysteine residues at positions which are well conserved [Schall et al.,supra; Loetscher et al., supra; Smith et al., supra; Nophar et al.,supra; Kohno et al., supra]. In TNFR1, the approximate boundaries of thefour CRDs are as follows: CRD1-amino acids 14 to about 53; CRD2-aminoacids from about 54 to about 97; CRD3-amino acids from about 98 to about138; CRD4-amino acids from about 139 to about 167. In TNFR2, CRD1includes amino acids 17 to about 54; CRD2-amino acids from about 55 toabout 97; CRD3-amino acids from about 98 to about 140; and CRD4-aminoacids from about 141 to about 179 [Banner et al., Cell, 73:431-435(1993)]. The potential role of the CRDs in ligand binding is alsodescribed by Banner et al., supra.

[0012] A similar repetitive pattern of CRDs exists in several othercell-surface proteins, including the p75 nerve growth factor receptor(NGFR) [Johnson et al., Cell, 47:545 (1986); Radeke et al., Nature,325:593 (1987)], the B cell antigen CD40 [Stamenkovic et al., EMBO J.,8:1403 (1989)], the T cell antigen OX40 [Mallet et al., EMBO J., 9:1063(1990)] and the Fas antigen [Yonehara et al., supra and Itoh et al.,supra]. CRDs are also found in the soluble TNFR (sTNFR)-like T2 proteinsof the Shope and myxoma poxviruses [Upton et al., Virology, 160:20-29(1987); Smith et al., Biochem. Biophys. Res. Commun., 176:335 (1991);Upton et al., Virology, 184:370 (1991)]. Optimal alignment of thesesequences indicates that the positions of the cysteine residues are wellconserved. These receptors are sometimes collectively referred to asmembers of the TNF/NGF receptor superfamily. Recent studies on p75NGFRshowed that the deletion of CRD1 [Welcher, A. A. et al., Proc. Natl.Acad. Sci. USA, 88:159-163 (1991)] or a 5-amino acid insertion in thisdomain [Yan, H. and Chao, M. V., J. Biol. Chem., 266:12099-12104 (1991)]had little or no effect on NGF binding [Yan, H. and Chao, M. V., supra].p75 NGFR contains a proline-rich stretch of about 60 amino acids,between its CRD4 and transmembrane region, which is not involved in NGFbinding [Peetre, C. et al., Eur. J. Hematol., 41:414-419 (1988);Seckinger, P. et al., J. Biol. Chem., 264:11966-11973 (1989); Yan, H.and Chao, M. V., supra]. A similar proline-rich region is found in TNFR2but not in TNFR1.

[0013] Itoh et al. disclose that the Apo-1 receptor can signal anapoptotic cell death similar to that signaled by the 55-kDa TNFR1 [Itohet al., supra]. Expression of the Apo-1 antigen has also been reportedto be down-regulated along with that of TNFR1 when cells are treatedwith either TNF-α or anti-Apo-1 mouse monoclonal antibody [Krammer etal., supra; Nagata et al., supra]. Accordingly, some investigators havehypothesized that cell lines that co-express both Apo-1 and TNFR1receptors may mediate cell killing through common signaling pathways[Id.].

[0014] The TNF family ligands identified to date, with the exception oflymphotoxin-α, are type II transmembrane proteins, whose C-terminus isextracellular. In contrast, the receptors in the TNF receptor (TNFR)family identified to date are type I transmembrane proteins. In both theTNF ligand and receptor families, however, homology identified betweenfamily members has been found mainly in the extracellular domain(“ECD”). Several of the TNF family cytokines, including TNF-α, Apo-1ligand and CD40 ligand, are cleaved proteolytically at the cell surface;the resulting protein in each case typically forms a homotrimericmolecule that functions as a soluble cytokine. TNF receptor familyproteins are also usually cleaved proteolytically to release solublereceptor ECDs that can function as inhibitors of the cognate cytokines.

[0015] Recently, other members of the mammalian TNFR family have beenidentified. In Marsters et al., Curr. Biol., 6:750 (1996), investigatorsdescribe a full length native sequence human polypeptide, called Apo-3,which exhibits similarity to the TNFR family in its extracellularcysteine-rich repeats and resembles TNFR1 and CD95 in that it contains acytoplasmic death domain sequence [see also Marsters et al., Curr.Biol., 6:1669 (1996)]. Apo-3 has also been referred to by otherinvestigators as DR3, wsl-1 and TRAMP [Chinnaiyan et al., Science,274:990 (1996); Kitson et al., Nature, 384:372 (1996); Bodmer et al.,Immunity, 6:79 (1997)].

[0016] Pan et al. have disclosed another TNF receptor family memberreferred to as “DR4” [Pan et al., Science, 276:111-113 (1997)]. The DR4was reported to contain a cytoplasmic death domain capable of engagingthe cell suicide apparatus. Pan et al. disclose that DR4 is believed tobe a receptor for the ligand known as Apo-2 ligand or TRAIL.

[0017] The Apoptosis-Inducing Signaling Complex

[0018] As presently understood, the cell death program contains at leastthree important elements—activators, inhibitors, and effectors; in C.elegans, these elements are encoded respectively by three genes, Ced-4,Ced-9 and Ced-3 [Steller, Science, 267:1445 (1995); Chinnaiyan et al.,Science, 275:1122-1126 (1997)]. Two of the TNFR family members, TNFR1and Fas/Apo1 (CD95), can activate apoptotic cell death [Chinnaiyan andDixit, Current Biology, 6:555-562 (1996); Fraser and Evan, Cell;85:781-784 (1996)]. TNFR1 is also known to mediate activation of thetranscription factor, NF-κB [Tartaglia et al., Cell, 74:845-853 (1993);Hsu et al., Cell, 84:299-308 (1996)]. In addition to some ECD homology,these two receptors share homology in their intracellular domain (ICD)in an oligomerization interface known as the death domain [Tartaglia etal., supra; Nagata, Cell, 88:355 (1997)]. Death domains are also foundin several metazoan proteins that regulate apoptosis, namely, theDrosophila protein, Reaper, and the mammalian proteins referred to asFADD/MORT1, TRADD, and RIP [Cleaveland and Ihle, Cell, 81:479-482(1995)]. Using the yeast-two hybrid system, Raven et al. report theidentification of protein, wsl-1, which binds to the TNFR1 death domain[Raven et al., Programmed Cell Death Meeting, Sep. 20-24, 1995, Abstractat page 127; Raven et al., European Cytokine Network, 7:Abstr. 82 atpage 210 (April-June 1996)]. The wsl-1 protein is described as beinghomologous to TNFR1 (48% identity) and having a restricted tissuedistribution. According to Raven et al., the tissue distribution ofwsl-1 is significantly different from the TNFR1 binding protein, TRADD.

[0019] Upon ligand binding and receptor clustering, TNFR1 and CD95 arebelieved to recruit FADD into a death-inducing signalling complex. CD95purportedly binds FADD directly, while TNFR1 binds FADD indirectly viaTRADD [Chinnaiyan et al., Cell, 81:505-512 (1995); Boldin et al., J.Biol. Chem., 270:387-391 (1995); Hsu et al., supra; Chinnaiyan et al.,J. Biol. Chem., 271:4961-4965 (1996)]. It has been reported that FADDserves as an adaptor protein which recruits the Ced-3-related protease,MACHα/FLICE (caspase 8), into the death signalling complex [Boldin etal., Cell, 85:803-815 (1996); Muzio et al., Cell, 85:817-827 (1996)].MACHα/FLICE appears to be the trigger that sets off a cascade ofapoptotic proteases, including the interleukin-1β converting enzyme(ICE) and CPP32/Yama, which may execute some critical aspects of thecell death programme [Fraser and Evan, supra].

[0020] It was recently disclosed that programmed cell death involves theactivity of members of a family of cysteine proteases related to the C.elegans cell death gene, ced-3, and to the mammalian IL-1-convertingenzyme, ICE. The activity of the ICE and CPP32/Yama proteases can beinhibited by the product of the cowpox virus gene, crmA [Ray et al.,Cell, 69:597-604 (1992); Tewari et al., Cell, 81:801-809 (1995)]. Recentstudies show that CrmA can inhibit TNFR1- and CD95-induced cell death[Enari et al., Nature, 375:78-81 (1995); Tewari et al., J. Biol. Chem.,270:3255-3260 (1995)].

[0021] As reviewed recently by Tewari et al., TNFR1, TNFR2 and CD40modulate the expression of proinflammatory and costimulatory cytokines,cytokine receptors, and cell adhesion molecules through activation ofthe transcription factor, NF-κB [Tewari et al., Curr. Op. Genet.Develop., 6:39-44 (1996)]. NF-κB is the prototype of a family of dimerictranscription factors whose subunits contain conserved Rel regions[Verma et al., Genes Develop., 9:2723-2735 (1996); Baldwin, Ann. Rev.Immunol., 14:649-681 (1996)]. In its latent form, NF-κB is complexedwith members of the IκB inhibitor family; upon inactivation of the IκBin response to certain stimuli, released NF-κB translocates to thenucleus where it binds to specific DNA sequences and activates genetranscription.

[0022] For a review of the TNF family of cytokines and their receptors,see Gruss and Dower, supra.

SUMMARY OF THE INVENTION

[0023] Applicants have identified cDNA clones that encode novelpolypeptides, designated in the present application as “Apo-2.” It isbelieved that Apo-2 is a member of the TNFR family; full-length nativesequence human Apo-2 polypeptide exhibits some similarities to someknown TNFRs, including a cytoplasmic death domain region. Full-lengthnative sequence human Apo-2 also exhibits similarity to the TNFR familyin its extracellular cysteine-rich repeats. Apo-2 polypeptide has beenfound to be capable of triggering caspase-dependent apoptosis andactivating NF-κB. Applicants surprisingly found that a solubleextracellular domain of Apo-2 binds Apo-2 ligand (“Apo-2L”) and caninhibit Apo-2 ligand function. It is presently believed that Apo-2ligand can signal via at least two different receptors, DR4 and thenewly described Apo-2 herein.

[0024] In one embodiment, the invention provides isolated Apo-2polypeptide. In particular, the invention provides isolated nativesequence Apo-2 polypeptide, which in one embodiment, includes an aminoacid sequence comprising residues 1 to 411 of FIG. 1 (SEQ ID NO:1). Inother embodiments, the isolated Apo-2 polypeptide comprises at leastabout 80% amino acid sequence identity with native sequence Apo-2polypeptide comprising residues 1 to 411 of FIG. 1 (SEQ ID NO:1).Optionally, the Apo-2 polypeptide is obtained or obtainable byexpressing the polypeptide encoded by the cDNA insert of the vectordeposited as ATCC 209021.

[0025] In another embodiment, the invention provides an isolatedextracellular domain (ECD) sequence of Apo-2. Optionally, the isolatedextracellular domain sequence comprises amino acid residues 54 to 182 ofFIG. 1 (SEQ ID NO:1).

[0026] In another embodiment, the invention provides an isolated deathdomain sequence of Apo-2. Optionally, the isolated death domain sequencecomprises amino acid residues 324 to 391 of FIG. 1 (SEQ ID NO:1).

[0027] In another embodiment, the invention provides chimeric moleculescomprising Apo-2 polypeptide fused to a heterologous polypeptide oramino acid sequence. An example of such a chimeric molecule comprises anApo-2 fused to an immunoglobulin sequence. Another example comprises anextracellular domain sequence of Apo-2 fused to a heterologouspolypeptide or amino acid sequence, such as an immunoglobulin sequence.

[0028] In another embodiment, the invention provides an isolated nucleicacid molecule encoding Apo-2 polypeptide. In one aspect, the nucleicacid molecule is RNA or DNA that encodes an Apo-2 polypeptide or aparticular domain of Apo-2, or is complementary to such encoding nucleicacid sequence, and remains stably bound to it under at least moderate,and optionally, under high stringency conditions. Such complementarynucleic acid may be fully complementary to the entire length of the RNAor DNA. It is contemplated that the complementary nucleic acid may alsobe complementary to only a fragment of the RNA or DNA nucleotidesequence. In one embodiment, the nucleic acid sequence is selected from:

[0029] (a) the coding region of the nucleic acid sequence of FIG. 1 (SEQID NO:2) that codes for residue 1 to residue 411 (i.e., nucleotides140-142 through 1370-1372), inclusive;

[0030] (b) the coding region of the nucleic acid sequence of FIG. 1 (SEQID NO:2) that codes for residue 1 to residue 182 (i.e., nucleotides140-142 through 683-685), inclusive;

[0031] (c) the coding region of the nucleic acid sequence of FIG. 1 (SEQID NO:2) that codes for residue 54 to residue 182 (i.e., nucleotides299-301 through 683-685), inclusive;

[0032] (d) the coding region of the nucleic acid sequence of FIG. 1 (SEQID NO:2) that codes for residue 324 to residue 391 (i.e., nucleotides1109-1111 through 1310-1312), inclusive; or

[0033] (e) a sequence corresponding to the sequence of (a), (b), (c) or(d) within the scope of degeneracy of the genetic code. The isolatednucleic acid may comprise the Apo-2 polypeptide cDNA insert of thevector deposited as ATCC 209021 which includes the nucleotide sequenceencoding Apo-2 polypeptide.

[0034] In a further embodiment, the invention provides a vectorcomprising the nucleic acid molecule encoding the Apo-2 polypeptide orparticular domain of Apo-2. A host cell comprising the vector or thenucleic acid molecule is also provided. A method of producing Apo-2 isfurther provided.

[0035] In another embodiment, the invention provides an antibody whichspecifically binds to Apo-2. The antibody may be an agonistic,antagonistic or neutralizing antibody. Single-chain antibodies anddimeric molecules, in particular homodimeric molecules, comprising Apo-2antibody are also provided.

[0036] In another embodiment, the invention provides non-human,transgenic or knock-out animals.

[0037] A further embodiment of the invention provides articles ofmanufacture and kits that include Apo-2 or Apo-2 antibodies.

BRIEF DESCRIPTION OF THE DRAWINGS

[0038]FIG. 1 shows the nucleotide sequence of a native sequence humanApo-2 cDNA (SEQ ID NO:2) and its derived amino acid sequence (SEQ IDNO:1).

[0039]FIG. 2A shows the derived amino acid sequence of a native sequencehuman Apo-2—the putative signal sequence is underlined, the putativetransmembrane domain is boxed, and the putative death domain sequence isdash underlined. The cysteines of the two cysteine-rich domains areindividually underlined.

[0040]FIG. 2B shows an alignment and comparison of the death domainsequences of native sequence human Apo-2, DR4, Apo-3/DR3, TNFR1, andFas/Apo-1 (CD95). Asterisks indicate residues that are essential fordeath signaling by TNFR1 [Tartaglia et al., supra].

[0041]FIG. 3 shows the interaction of the Apo-2 ECD with Apo-2L.Supernatants from mock-transfected 293 cells or from 293 cellstransfected with Flag epitope-tagged Apo-2 ECD were incubated withpoly-His-tagged Apo-2L and subjected to immunoprecipitation withanti-Flag conjugated or Nickel conjugated agarose beads. Theprecipitated proteins were resolved by electrophoresis on polyacrylamidegels, and detected by immunoblot with anti-Apo-2L or anti-Flag antibody.

[0042]FIG. 4 shows the induction of apoptosis by Apo-2 and inhibition ofApo-2L activity by soluble Apo-2 ECD. Human 293 cells (A, B) or HeLacells (C) were transfected by pRK5 vector or by pRK5-based plasmidsencoding Apo-2 and/or CrmA. Apoptosis was assessed by morphology (A),DNA fragmentation (B), or by FACS (C-E). Soluble Apo-2L waspre-incubated with buffer or affinity-purified Apo-2 ECD together withanti-Flag antibody or Apo-2 ECD immunoadhesin or DR4 or TNFR1immunoadhesins and added to HeLa cells. The cells were later analyzedfor apoptosis (D). Dose-response analysis using Apo-2L with Apo-2 ECDimmunoadhesin was also determined (E).

[0043]FIG. 5 shows activation of NF-κB by Apo-2, DR4, and Apo-2L. (A)HeLa cells were transfected with expression plasmids encoding theindicated proteins. Nuclear extracts were prepared and analyzed by anelectrophoretic mobility shift assay. (B) HeLa cells or MCF7 cells weretreated with buffer, Apo-2L or TNF-alpha and assayed for NF-κB activity.(C) HeLa cells were preincubated with buffer, ALLN or cyclohexamidebefore addition of Apo-2L. Apoptosis was later analyzed by FACS.

[0044]FIG. 6A shows expression of Apo-2 mRNA in human tissues asanalyzed by Northern hybridization of human tissue poly A RNA blots.

[0045]FIG. 6B shows expression of Apo-2 mRNA in human cancer cell linesas analyzed by Northern hybridization of human cancer cell line poly ARNA blots.

[0046]FIG. 7 shows the FACS analysis of an Apo-2 antibody, 3F11.39.7(illustrated by the bold lines) as compared to IgG controls (dottedlines). The 3F11.39.7 antibody recognized the Apo-2 receptor expressedin human 9D cells.

[0047]FIG. 8 is a graph showing percent (%) apoptosis induced in 9Dcells by Apo-2 antibody 3F11.39.7, in the absence of goat anti-mouse IgGFc.

[0048]FIG. 9 is a bar diagram showing percent (%) apoptosis, as comparedto Apo-2L, in 9D cells by Apo-2 antibody 3F11.39.7 in the presence orabsence of goat anti-mouse IgG Fc.

[0049]FIG. 10 is a bar diagram illustrating the ability of Apo-2antibody 3F11.39.7 to block the apoptosis induced by Apo-2L in 9D cells.

[0050]FIG. 11 is a graph showing results of an ELISA testing binding ofApo-2 antibody 3F11.39.7 to Apo-2 and to other known Apo-2L receptorsreferred to as DR4, DcR1, and DcR2.

[0051]FIG. 12A is a graph showing the results of an ELISA assayevaluating binding of the 16E2 antibody to Apo-2, DR4, DcR1, DcR2 andCD4-Ig.

[0052]FIG. 12B is a graph showing the results of an ELISA assayevaluating binding of the 20E6 antibody to Apo-2, DR4, DcR1, DcR2 andCD4-Ig.

[0053]FIG. 12C is a graph showing the results of an ELISA assayevaluating binding of the 24C4 antibody to Apo-2, DR4, DcR1, DcR2 andCD4-Ig.

[0054]FIG. 13A is a graph showing agonistic activity of the 16E2antibody, as compared to Apo-2L, in an apoptosis assay (crystal violetstain) using SK-MES-1 cells.

[0055]FIG. 13B is a bar diagram showing agonistic activity of the 16E2antibody, as compared to 7D5 scFv antibody (an anti-tissue factorantibody), in an apoptosis assay (crystal violet stain) using SK-MES-1cells.

[0056]FIG. 13C is a bar diagram showing agonistic activity of the 16E2antibody, as compared to 7D5 scFv antibody, in an apoptosis assay(annexin V-biotin/streptavidin-[S³⁵]) using SK-MES-1 cells.

[0057]FIG. 14A is a graph showing agonistic activity of the 20E6antibody, as compared to Apo-2L, in an apoptosis assay (crystal violetstain) using SK-MES-1 cells.

[0058]FIG. 14B is a graph showing agonistic activity of the 20E6antibody by a comparison between results obtained in the crystal violetand annexin V-biotin/streptavidin-[S³⁵] apoptosis assays.

[0059]FIG. 14C is a graph showing agonistic activity of gD-tagged 16E2antibody, as compared to Apo-2L, in an apoptosis assay (crystal violetstain) using SK-MES-1 cells

[0060]FIG. 15A shows the nucleotide sequence of the single chainantibody (scFv) fragment referred to as 16E2 (SEQ ID NO:6).

[0061]FIG. 15B shows the nucleotide sequence of the single chainantibody (scFv) fragment referred to as 20E6 (SEQ ID NO:7).

[0062]FIG. 15C shows the nucleotide sequence of the single chainantibody (scFv) fragment referred to as 24C4 (SEQ ID NO:8).

[0063]FIG. 16 shows the single chain antibody (scFv) fragments referredto as 16E2, 20E6 and 24C4, with the respective amino acid sequences forthe signal sequence and the heavy and light chain CDR regions identified(CDR1, CDR2, and CDR3 regions are underlined).

DETAILED DESCRIPTION OF THE PREFERRED EMBODIMENTS

[0064] I. Definitions

[0065] The terms “Apo-2 polypeptide” and “Apo-2” when used hereinencompass native sequence Apo-2 and Apo-2 variants (which are furtherdefined herein). These terms encompass Apo-2 from a variety of mammals,including humans. The Apo-2 may be isolated from a variety of sources,such as from human tissue types or from another source, or prepared byrecombinant or synthetic methods.

[0066] A “native sequence Apo-2” comprises a polypeptide having the sameamino acid sequence as an Apo-2 derived from nature. Thus, a nativesequence Apo-2 can have the amino acid sequence of naturally-occurringApo-2 from any mammal. Such native sequence Apo-2 can be isolated fromnature or can be produced by recombinant or synthetic means. The term“native sequence Apo-2” specifically encompasses naturally-occurringtruncated or secreted forms of the Apo-2 (e.g., an extracellular domainsequence), naturally-occurring variant forms (e.g., alternativelyspliced forms) and naturally-occurring allelic variants of the Apo-2. Anaturally-occurring variant form of the Apo-2 includes an Apo-2 havingan amino acid substitution at residue 410 in the amino acid sequenceshown in FIG. 1 (SEQ ID NO:1). In one embodiment of suchnaturally-occurring variant form, the leucine residue at position 410 issubstituted by a methionine residue. In FIG. 1 (SEQ ID NO:1), the aminoacid residue at position 410 is identified as “Xaa” to indicate that theamino acid may, optionally, be either leucine or methionine. In FIG. 1(SEQ ID NO:2), the nucleotide at position 1367 is identified as “W” toindicate that the nucleotide may be either adenine (A) or thymine (T) oruracil (U). In one embodiment of the invention, the native sequenceApo-2 is a mature or full-length native sequence Apo-2 comprising aminoacids 1 to 411 of FIG. 1 (SEQ ID NO:1). Optionally, the Apo-2 isobtained or obtainable by expressing the polypeptide encoded by the cDNAinsert of the vector deposited as ATCC 209021.

[0067] The “Apo-2 extracellular domain” or “Apo-2 ECD” refers to a formof Apo-2 which is essentially free of the transmembrane and cytoplasmicdomains of Apo-2. Ordinarily, Apo-2 ECD will have less than 1% of suchtransmembrane and/or cytoplasmic domains and preferably, will have lessthan 0.5% of such domains. Optionally, Apo-2 ECD will comprise aminoacid residues 54 to 182 of FIG. 1 (SEQ ID NO:1) or amino acid residues 1to 182 of FIG. 1 (SEQ ID NO:1). Optionally, Apo-2 ECD will comprise oneor more cysteine-rich domains, and preferably, one or both of thecysteine-rich domains identified herein (see FIG. 2A). It will beunderstood by the skilled artisan that the transmembrane domainidentified for the Apo-2 polypeptide herein is identified pursuant tocriteria routinely employed in the art for identifying that type ofhydrophobic domain. The exact boundaries of a transmembrane domain mayvary but most likely by no more than about 5 amino acids at either endof the domain specifically mentioned herein.

[0068] “Apo-2 variant” means a biologically active Apo-2 as definedbelow having at least about 80% amino acid sequence identity with theApo-2 having the deduced amino acid sequence shown in FIG. 1 (SEQ IDNO:1) for a full-length native sequence human Apo-2 or the sequencesidentified herein for Apo-2 ECD or death domain. Such Apo-2 variantsinclude, for instance, Apo-2 polypeptides wherein one or more amino acidresidues are added, or deleted, at the N- or C-terminus of the sequenceof FIG. 1 (SEQ ID NO:1) or the sequences identified herein for Apo-2 ECDor death domain. Ordinarily, an Apo-2 variant will have at least about80% amino acid sequence identity, more preferably at least about 90%amino acid sequence identity, and even more preferably at least about95% amino acid sequence identity with the amino acid sequence of FIG. 1(SEQ ID NO:1) or the sequences identified herein for Apo-2 ECD or deathdomain.

[0069] “Percent (%) amino acid sequence identity” with respect to theApo-2 sequences identified herein is defined as the percentage of aminoacid residues in a candidate sequence that are identical with the aminoacid residues in the Apo-2 sequence, after aligning the sequences andintroducing gaps, if necessary, to achieve the maximum percent sequenceidentity, and not considering any conservative substitutions as part ofthe sequence identity. Alignment for purposes of determining percentamino acid sequence identity can be achieved in various ways that arewithin the skill in the art, for instance, using publicly availablecomputer software such as ALIGN™ or Megalign (DNASTAR) software. Thoseskilled in the art can determine appropriate parameters for measuringalignment, including any algorithms needed to achieve maximal alignmentover the full length of the sequences being compared.

[0070] The term “epitope tagged” when used herein refers to a chimericpolypeptide comprising Apo-2 or Apo-2 antibody, or a domain sequencethereof, fused to a “tag polypeptide”. The tag polypeptide has enoughresidues to provide an epitope against which an antibody can be made,yet is short enough such that it does not interfere with activity of theApo-2 or Apo-2 antibody. The tag polypeptide preferably also is fairlyunique so that the antibody does not substantially cross-react withother epitopes. Suitable tag polypeptides generally have at least sixamino acid residues and usually between about 8 to about 50 amino acidresidues (preferably, between about 10 to about 20 residues).

[0071] “Isolated,” when used to describe the various polypeptidesdisclosed herein, means polypeptide that has been identified andseparated and/or recovered from a component of its natural environment.Contaminant components of its natural environment are materials thatwould typically interfere with diagnostic or therapeutic uses for thepolypeptide, and may include enzymes, hormones, and other proteinaceousor non-proteinaceous solutes. In preferred embodiments, the polypeptidewill be purified (1) to a degree sufficient to obtain at least 15residues of N-terminal or internal amino acid sequence by use of aspinning cup sequenator, or (2) to homogeneity by SDS-PAGE undernon-reducing or reducing conditions using Coomassie blue or, preferably,silver stain. Isolated polypeptide includes polypeptide in situ withinrecombinant cells, since at least one component of the Apo-2 naturalenvironment will not be present. Ordinarily, however, isolatedpolypeptide will be prepared by at least one purification step.

[0072] An “isolated” Apo-2 nucleic acid molecule is a nucleic acidmolecule that is identified and separated from at least one contaminantnucleic acid molecule with which it is ordinarily associated in thenatural source of the Apo-2 nucleic acid. An isolated Apo-2 nucleic acidmolecule is other than in the form or setting in which it is found innature. Isolated Apo-2 nucleic acid molecules therefore aredistinguished from the Apo-2 nucleic acid molecule as it exists innatural cells. However, an isolated Apo-2 nucleic acid molecule includesApo-2 nucleic acid molecules contained in cells that ordinarily expressApo-2 where, for example, the nucleic acid molecule is in a chromosomallocation different from that of natural cells.

[0073] The term “control sequences” refers to DNA sequences necessaryfor the expression of an operably linked coding sequence in a particularhost organism. The control sequences that are suitable for prokaryotes,for example, include a promoter, optionally an operator sequence, and aribosome binding site. Eukaryotic cells are known to utilize promoters,polyadenylation signals, and enhancers.

[0074] Nucleic acid is “operably linked” when it is placed into afunctional relationship with another nucleic acid sequence. For example,DNA for a presequence or secretory leader is operably linked to DNA fora polypeptide if it is expressed as a preprotein that participates inthe secretion of the polypeptide; a promoter or enhancer is operablylinked to a coding sequence if it affects the transcription of thesequence; or a ribosome binding site is operably linked to a codingsequence if it is positioned so as to facilitate translation. Generally,“operably linked” means that the DNA sequences being linked arecontiguous, and, in the case of a secretory leader, contiguous and inreading phase. However, enhancers do not have to be contiguous. Linkingis accomplished by ligation at convenient restriction sites. If suchsites do not exist, the synthetic oligonucleotide adaptors or linkersare used in accordance with conventional practice.

[0075] The term “antibody” is used in the broadest sense andspecifically covers anti-Apo-2 monoclonal antibodies (including agonist,antagonist, and blocking or neutralizing antibodies) and anti-Apo-2antibody compositions with polyepitopic specificity.

[0076] The term “monoclonal antibody” as used herein refers to anantibody obtained from a population of substantially homogeneousantibodies, i.e., the individual antibodies comprising the populationare identical except for possible naturally-occurring mutations that maybe present in minor amounts. Monoclonal antibodies are highly specific,being directed against a single antigenic site. Furthermore, in contrastto conventional (polyclonal) antibody preparations which typicallyinclude different antibodies directed against different determinants(epitopes), each monoclonal antibody is directed against a singledeterminant on the antigen.

[0077] The monoclonal antibodies herein include hybrid and recombinantantibodies produced by splicing a variable (including hypervariable)domain of an anti-Apo-2 antibody with a constant domain, or a lightchain with a heavy chain, or a chain from one species with a chain fromanother species, or fusions with heterologous proteins, regardless ofspecies of origin or immunoglobulin class or subclass designation, aswell as antibody fragments (e.g., Fab, F(ab′)₂, and Fv), so long as theyexhibit the desired biological activity. See, e.g. U.S. Pat. No.4,816,567 and Mage et al., in Monoclonal Antibody Production Techniquesand Applications, pp.79-97 (Marcel Dekker, Inc.: New York, 1987).

[0078] Thus, the modifier “monoclonal” indicates the character of theantibody as being obtained from a substantially homogeneous populationof antibodies, and is not to be construed as requiring production of theantibody by any particular method. For example, the monoclonalantibodies to be used in accordance with the present invention may bemade by the hybridoma method first described by Kohler and Milstein,Nature, 256:495 (1975), or may be made by recombinant DNA methods suchas described in U.S. Pat. No. 4,816,567. The “monoclonal antibodies” mayalso be isolated from phage libraries generated using the techniquesdescribed in McCafferty et al., Nature, 348:552-554 (1990), for example.

[0079] “Single-chain Fv” or “scFv” antibody fragments comprise the V_(H)and V_(L) domains of antibody, wherein these domains are present in asingle polypeptide chain. Generally, the Fv polypeptide furthercomprises a polypeptide linker between the V_(H) and V_(L) domains whichenables the scFv to form the desired structure for antigen binding. Fora review of scFv see, e.g., Pluckthun, The Pharmacology of MonoclonalAntibodies, vol. 113, Rosenburg and Moore eds. Springer-Verlag, NewYork, pp. 269-315 (1994). The scFv antibody fragments of the presentinvention include but are not limited to the 16E2, 20E6 and 24C4antibodies described in detail below. Within the scope of the scFvantibodies of the invention are scFv antibodies comprising VH and VLdomains that include one or more of the CDR regions identified for the16E2, 20E6 and 24C4 antibodies.

[0080] “Humanized” forms of non-human (e.g. murine) antibodies arespecific chimeric immunoglobulins, immunoglobulin chains, or fragmentsthereof (such as Fv, Fab, Fab′, F(ab′)₂ or other antigen-bindingsubsequences of antibodies) which contain minimal sequence derived fromnon-human immunoglobulin. For the most part, humanized antibodies arehuman immunoglobulins (recipient antibody) in which residues from acomplementary determining region (CDR) of the recipient are replaced byresidues from a CDR of a non-human species (donor antibody) such asmouse, rat, or rabbit having the desired specificity, affinity, andcapacity. In some instances, Fv framework region (FR) residues of thehuman immunoglobulin are replaced by corresponding non-human residues.Furthermore, the humanized antibody may comprise residues which arefound neither in the recipient antibody nor in the imported CDR orframework sequences. These modifications are made to further refine andoptimize antibody performance. In general, the humanized antibody willcomprise substantially all of at least one, and typically two, variabledomains, in which all or substantially all of the CDR regions correspondto those of a non-human immunoglobulin and all or substantially all ofthe FR regions are those of a human immunoglobulin consensus sequence.The humanized antibody optimally also will comprise at least a portionof an immunoglobulin constant region or domain (Fc), typically that of ahuman immunoglobulin.

[0081] “Biologically active” and “desired biological activity” for thepurposes herein means (1) having the ability to modulate apoptosis(either in an agonistic or stimulating manner or in an antagonistic orblocking manner) in at least one type of mammalian cell in vivo or exvivo; (2) having the ability to bind Apo-2 ligand; or (3) having theability to modulate Apo-2 ligand signaling and Apo-2 ligand activity.

[0082] The terms “apoptosis” and “apoptotic activity” are used in abroad sense and refer to the orderly or controlled form of cell death inmammals that is typically accompanied by one or more characteristic cellchanges, including condensation of cytoplasm, loss of plasma membranemicrovilli, segmentation of the nucleus, degradation of chromosomal DNAor loss of mitochondrial function. This activity can be determined andmeasured, for instance, by cell viability assays, FACS analysis or DNAelectrophoresis, all of which are known in the art.

[0083] The terms “treating,” “treatment,” and “therapy” as used hereinrefer to curative therapy, prophylactic therapy, and preventativetherapy.

[0084] The terms “cancer” and “cancerous” refer to or describe thephysiological condition in mammals that is typically characterized byunregulated cell growth. Examples of cancer include but are not limitedto, carcinoma, lymphoma, blastoma, sarcoma, and leukemia. Moreparticular examples of such cancers include squamous cell cancer,small-cell lung cancer, non-small cell lung cancer, blastoma,gastrointestinal cancer, renal cancer, pancreatic cancer, glioblastoma,neuroblastoma, cervical cancer, ovarian cancer, liver cancer, stomachcancer, bladder cancer, hepatoma, breast cancer, colon cancer,colorectal cancer, endometrial carcinoma, salivary gland carcinoma,kidney cancer, liver cancer, prostate cancer, vulval cancer, thyroidcancer, hepatic carcinoma and various types of head and neck cancer.

[0085] The term “mammal” as used herein refers to any mammal classifiedas a mammal, including humans, cows, horses, dogs and cats. In apreferred embodiment of the invention, the mammal is a human.

[0086] II. Compositions and Methods of the Invention

[0087] The present invention provides newly identified and isolatedApo-2 polypeptides and Apo-2 antibodies. In particular, Applicants haveidentified and isolated various human Apo-2 polypeptides. The propertiesand characteristics of some of these Apo-2 polypeptides and anti-Apo-2antibodies are described in further detail in the Examples below. Basedupon the properties and characteristics of the Apo-2 polypeptidesdisclosed herein, it is Applicants' present belief that Apo-2 is amember of the TNFR family.

[0088] A description follows as to how Apo-2, as well as Apo-2 chimericmolecules and anti-Apo-2 antibodies, may be prepared.

[0089] A. Preparation of Apo-2

[0090] The description below relates primarily to production of Apo-2 byculturing cells transformed or transfected with a vector containingApo-2 nucleic acid. It is of course, contemplated that alternativemethods, which are well known in the art, may be employed to prepareApo-2.

[0091] 1. Isolation of DNA Encoding Apo-2

[0092] The DNA encoding Apo-2 may be obtained from any cDNA libraryprepared from tissue believed to possess the Apo-2 mRNA and to expressit at a detectable level. Accordingly, human Apo-2 DNA can beconveniently obtained from a cDNA library prepared from human tissues,such as the bacteriophage libraries of human pancreas and kidney cDNAdescribed in Example 1. The Apo-2-encoding gene may also be obtainedfrom a genomic library or by oligonucleotide synthesis.

[0093] Libraries can be screened with probes (such as antibodies to theApo-2 or oligonucleotides of at least about 20-80 bases) designed toidentify the gene of interest or the protein encoded by it. Screeningthe cDNA or genomic library with the selected probe may be conductedusing standard procedures, such as described in Sambrook et al.,Molecular Cloning: A Laboratory Manual (New York: Cold Spring HarborLaboratory Press, 1989). An alternative means to isolate the geneencoding Apo-2 is to use PCR methodology [Sambrook et al., supra;Dieffenbach et al., PCR Primer:A Laboratory Manual (Cold Spring HarborLaboratory Press, 1995)].

[0094] A preferred method of screening employs selected oligonucleotidesequences to screen cDNA libraries from various human tissues. Example 1below describes techniques for screening a cDNA library. Theoligonucleotide sequences selected as probes should be of sufficientlength and sufficiently unambiguous that false positives are minimized.The oligonucleotide is preferably labeled such that it can be detectedupon hybridization to DNA in the library being screened. Methods oflabeling are well known in the art, and include the use of radiolabelslike ³²P-labeled ATP, biotinylation or enzyme labeling. Hybridizationconditions, including moderate stringency and high stringency, areprovided in Sambrook et al., supra.

[0095] Nucleic acid having all the protein coding sequence may beobtained by screening selected cDNA or genomic libraries using thededuced amino acid sequence disclosed herein for the first time, and, ifnecessary, using conventional primer extension procedures as describedin Sambrook et al., supra, to detect precursors and processingintermediates of mRNA that may not have been reverse-transcribed intocDNA.

[0096] Apo-2 variants can be prepared by introducing appropriatenucleotide changes into the Apo-2 DNA, or by synthesis of the desiredApo-2 polypeptide. Those skilled in the art will appreciate that aminoacid changes may alter post-translational processes of the Apo-2, suchas changing the number or position of glycosylation sites or alteringthe membrane anchoring characteristics.

[0097] Variations in the native full-length sequence Apo-2 or in variousdomains of the Apo-2 described herein, can be made, for example, usingany of the techniques and guidelines for conservative andnon-conservative mutations set forth, for instance, in U.S. Pat. No.5,364,934. Variations may be a substitution, deletion or insertion ofone or more codons encoding the Apo-2 that results in a change in theamino acid sequence of the Apo-2 as compared with the native sequenceApo-2. Optionally the variation is by substitution of at least one aminoacid with any other amino acid in one or more of the domains of theApo-2 molecule. The variations can be made using methods known in theart such as oligonucleotide-mediated (site-directed) mutagenesis,alanine scanning, and PCR mutagenesis. Site-directed mutagenesis [Carteret al., Nucl. Acids Res., 13:4331 (1986); Zoller et al., Nucl. AcidsRes., 10:6487 (1987)], cassette mutagenesis [Wells et al., Gene, 34:315(1985)], restriction selection mutagenesis [Wells et al., Philos. Trans.R. Soc. London SerA, 317:415 (1986)] or other known techniques can beperformed on the cloned DNA to produce the Apo-2 variant DNA.

[0098] Scanning amino acid analysis can also be employed to identify oneor more amino acids along a contiguous sequence which are involved inthe interaction with a particular ligand or receptor. Among thepreferred scanning amino acids are relatively small, neutral aminoacids. Such amino acids include alanine, glycine, serine, and cysteine.Alanine is the preferred scanning amino acid among this group because iteliminates the side-chain beyond the beta-carbon and is less likely toalter the main-chain conformation of the variant. Alanine is alsopreferred because it is the most common amino acid. Further, it isfrequently found in both buried and exposed positions [Creighton, TheProteins, (W.H. Freeman & Co., N.Y.); Chothia, J. Mol. Biol., 150:1(1976)]. If alanine substitution does not yield adequate amounts ofvariant, an isoteric amino acid can be used.

[0099] Once selected Apo-2 variants are produced, they can be contactedwith, for instance, Apo-2L, and the interaction, if any, can bedetermined. The interaction between the Apo-2 variant and Apo-2L can bemeasured by an in vitro assay, such as described in the Examples below.While any number of analytical measurements can be used to compareactivities and properties between a native sequence Apo-2 and an Apo-2variant, a convenient one for binding is the dissociation constant K_(d)of the complex formed between the Apo-2 variant and Apo-2L as comparedto the K_(d) for the native sequence Apo-2. Generally, a ≧3-foldincrease or decrease in K_(d) per substituted residue indicates that thesubstituted residue(s) is active in the interaction of the nativesequence Apo-2 with the Apo-2L.

[0100] Optionally, representative sites in the Apo-2 sequence suitablefor mutagenesis would include sites within the extracellular domain, andparticularly, within one or both of the cysteine-rich domains. Suchvariations can be accomplished using the methods described above.

[0101] 2. Insertion of Nucleic Acid into a Replicable Vector

[0102] The nucleic acid (e.g., cDNA or genomic DNA) encoding Apo-2 maybe inserted into a replicable vector for further cloning (amplificationof the DNA) or for expression. Various vectors are publicly available.The vector components generally include, but are not limited to, one ormore of the following: a signal sequence, an origin of replication, oneor more marker genes, an enhancer element, a promoter, and atranscription termination sequence, each of which is described below.

[0103] (i) Signal Sequence Component

[0104] The Apo-2 may be produced recombinantly not only directly, butalso as a fusion polypeptide with a heterologous polypeptide, which maybe a signal sequence or other polypeptide having a specific cleavagesite at the N-terminus of the mature protein or polypeptide. In general,the signal sequence may be a component of the vector, or it may be apart of the Apo-2 DNA that is inserted into the vector. The heterologoussignal sequence selected preferably is one that is recognized andprocessed (i.e., cleaved by a signal peptidase) by the host cell. Thesignal sequence may be a prokaryotic signal sequence selected, forexample, from the group of the alkaline phosphatase, penicillinase, lpp,or heat-stable enterotoxin IT leaders. For yeast secretion the signalsequence may be, e.g., the yeast invertase leader, alpha factor leader(including Saccharomyces and Kluyveromyces α-factor leaders, the latterdescribed in U.S. Pat. No. 5,010,182), or acid phosphatase leader, theC. albicans glucoamylase leader (EP 362,179 published Apr. 4, 1990), orthe signal described in WO 90/13646 published Nov. 15, 1990. Inmammalian cell expression the native Apo-2 presequence that normallydirects insertion of Apo-2 in the cell membrane of human cells in vivois satisfactory, although other mammalian signal sequences may be usedto direct secretion of the protein, such as signal sequences fromsecreted polypeptides of the same or related species, as well as viralsecretory leaders, for example, the herpes simplex glycoprotein Dsignal.

[0105] The DNA for such precursor region is preferably ligated inreading frame to DNA encoding Apo-2.

[0106] (ii) Origin of Replication Component

[0107] Both expression and cloning vectors contain a nucleic acidsequence that enables the vector to replicate in one or more selectedhost cells. Generally, in cloning vectors this sequence is one thatenables the vector to replicate independently of the host chromosomalDNA, and includes origins of replication or autonomously replicatingsequences. Such sequences are well known for a variety of bacteria,yeast, and viruses. The origin of replication from the plasmid pBR322 issuitable for most Gram-negative bacteria, the 2μ plasmid origin issuitable for yeast, and various viral origins (SV40, polyoma,adenovirus, VSV or BPV) are useful for cloning vectors in mammaliancells. Generally, the origin of replication component is not needed formammalian expression vectors (the SV40 origin may typically be usedbecause it contains the early promoter).

[0108] Most expression vectors are “shuttle” vectors, i.e., they arecapable of replication in at least one class of organisms but can betransfected into another organism for expression. For example, a vectoris cloned in E. coli and then the same vector is transfected into yeastor mammalian cells for expression even though it is not capable ofreplicating independently of the host cell chromosome.

[0109] DNA may also be amplified by insertion into the host genome. Thisis readily accomplished using Bacillus species as hosts, for example, byincluding in the vector a DNA sequence that is complementary to asequence found in Bacillus genomic DNA. Transfection of Bacillus withthis vector results in homologous recombination with the genome andinsertion of Apo-2 DNA. However, the recovery of genomic DNA encodingApo-2 is more complex than that of an exogenously replicated vectorbecause restriction enzyme digestion is required to excise the Apo-2DNA.

[0110] (iii) Selection Gene Component

[0111] Expression and cloning vectors typically contain a selectiongene, also termed a selectable marker. This gene encodes a proteinnecessary for the survival or growth of transformed host cells grown ina selective culture medium. Host cells not transformed with the vectorcontaining the selection gene will not survive in the culture medium.Typical selection genes encode proteins that (a) confer resistance toantibiotics or other toxins, e.g., ampicillin, neomycin, methotrexate,or tetracycline, (b) complement auxotrophic deficiencies, or (c) supplycritical nutrients not available from complex media, e.g., the geneencoding D-alanine racemase for Bacilli.

[0112] One example of a selection scheme utilizes a drug to arrestgrowth of a host cell. Those cells that are successfully transformedwith a heterologous gene produce a protein conferring drug resistanceand thus survive the selection regimen. Examples of such dominantselection use the drugs neomycin [Southern et al., J. Molec. Appl.Genet., 1:327 (1982)], mycophenolic acid (Mulligan et al., Science,209:1422 (1980)) or hygromycin [Sugden et al., Mol. Cell. Biol.,5:410-413 (1985)]. The three examples given above employ bacterial genesunder eukaryotic control to convey resistance to the appropriate drugG418 or neomycin (geneticin), xgpt (mycophenolic acid), or hygromycin,respectively.

[0113] Another example of suitable selectable markers for mammaliancells are those that enable the identification of cells competent totake up the Apo-2 nucleic acid, such as DHFR or thymidine kinase. Themammalian cell transformants are placed under selection pressure thatonly the transformants are uniquely adapted to survive by virtue ofhaving taken up the marker. Selection pressure is imposed by culturingthe transformants under conditions in which the concentration ofselection agent in the medium is successively changed, thereby leadingto amplification of both the selection gene and the DNA that encodesApo-2. Amplification is the process by which genes in greater demand forthe production of a protein critical for growth are reiterated in tandemwithin the chromosomes of successive generations of recombinant cells.Increased quantities of Apo-2 are synthesized from the amplified DNA.Other examples of amplifiable genes include metallothionein-I and -II,adenosine deaminase, and ornithine decarboxylase.

[0114] Cells transformed with the DHFR selection gene may first beidentified by culturing all of the transformants in a culture mediumthat contains methotrexate (Mtx), a competitive antagonist of DHFR. Anappropriate host cell when wild-type DHFR is employed is the Chinesehamster ovary (CHO) cell line deficient in DHFR activity, prepared andpropagated as described by Urlaub et al., Proc. Natl. Acad. Sci. USA,77:4216 (1980). The transformed cells are then exposed to increasedlevels of methotrexate. This leads to the synthesis of multiple copiesof the DHFR gene, and, concomitantly, multiple copies of other DNAcomprising the expression vectors, such as the DNA encoding Apo-2. Thisamplification technique can be used with any otherwise suitable host,e.g., ATCC No. CCL61 CHO-K1, notwithstanding the presence of endogenousDHFR if, for example, a mutant DHFR gene that is highly resistant to Mtxis employed (EP 117,060).

[0115] Alternatively, host cells (particularly wild-type hosts thatcontain endogenous DHFR) transformed or co-transformed with DNAsequences encoding Apo-2, wild-type DHFR protein, and another selectablemarker such as aminoglycoside 3′-phosphotransferase (APH) can beselected by cell growth in medium containing a selection agent for theselectable marker such as an aminoglycosidic antibiotic, e.g.,kanamycin, neomycin, or G418. See U.S. Pat. No. 4,965,199.

[0116] A suitable selection gene for use in yeast is the trp1 genepresent in the yeast plasmid YRp7 [Stinchcomb et al., Nature, 282:39(1979); Kingsman et al., Gene, 7:141 (1979); Tschemper et al., Gene,10:157 (1980)]. The trp1 gene provides a selection marker for a mutantstrain of yeast lacking the ability to grow in tryptophan, for example,ATCC No. 44076 or PEP4-1 [Jones, Genetics, 85:12 (1977)]. The presenceof the trp1 lesion in the yeast host cell genome then provides aneffective environment for detecting transformation by growth in theabsence of tryptophan. Similarly, Leu2-deficient yeast strains (ATCC20,622 or 38,626) are complemented by known plasmids bearing the Leu2gene.

[0117] In addition, vectors derived from the 1.6 μm circular plasmidpKD1 can be used for transformation of Kluyveromyces yeasts [Bianchi etal., Curr. Genet., 12:185 (1987)]. More recently, an expression systemfor large-scale production of recombinant calf chymosin was reported forK. lactis [Van den Berg, Bio/Technology, 8:135 (1990)]. Stablemulti-copy expression vectors for secretion of mature recombinant humanserum albumin by industrial strains of Kluyveromyces have also beendisclosed [Fleer et al., Bio/Technology, 9:968-975 (1991)].

[0118] (iv) Promoter Component

[0119] Expression and cloning vectors usually contain a promoter that isrecognized by the host organism and is operably linked to the Apo-2nucleic acid sequence. Promoters are untranslated sequences locatedupstream (5′) to the start codon of a structural gene (generally withinabout 100 to 1000 bp) that control the transcription and translation ofparticular nucleic acid sequence, such as the Apo-2 nucleic acidsequence, to which they are operably linked. Such promoters typicallyfall into two classes, inducible and constitutive. Inducible promotersare promoters that initiate increased levels of transcription from DNAunder their control in response to some change in culture conditions,e.g., the presence or absence of a nutrient or a change in temperature.At this time a large number of promoters recognized by a variety ofpotential host cells are well known. These promoters are operably linkedto Apo-2 encoding DNA by removing the promoter from the source DNA byrestriction enzyme digestion and inserting the isolated promotersequence into the vector. Both the native Apo-2 promoter sequence andmany heterologous promoters may be used to direct amplification and/orexpression of the Apo-2 DNA.

[0120] Promoters suitable for use with prokaryotic hosts include theβ-lactamase and lactose promoter systems [Chang et al., Nature, 275:615(1978); Goeddel et al., Nature, 281:544 (1979)], alkaline phosphatase, atryptophan (trp) promoter system [Goeddel, Nucleic Acids Res., 8:4057(1980); EP 36,776], and hybrid promoters such as the tac promoter[deBoer et al., Proc. Natl. Acad. Sci. USA, 80:21-25 (1983)]. However,other known bacterial promoters are suitable. Their nucleotide sequenceshave been published, thereby enabling a skilled worker operably toligate them to DNA encoding Apo-2 [Siebenlist et al., Cell, 20:269(1980)] using linkers or adaptors to supply any required restrictionsites. Promoters for use in bacterial systems also will contain aShine-Dalgarno (S.D.) sequence operably linked to the DNA encodingApo-2.

[0121] Promoter sequences are known for eukaryotes. Virtually alleukaryotic genes have an AT-rich region located approximately 25 to 30bases upstream from the site where transcription is initiated. Anothersequence found 70 to 80 bases upstream from the start of transcriptionof many genes is a CXCAAT region where X may be any nucleotide. At the3′ end of most eukaryotic genes is an AATAAA sequence that may be thesignal for addition of the poly A tail to the 3′ end of the codingsequence. All of these sequences are suitably inserted into eukaryoticexpression vectors.

[0122] Examples of suitable promoting sequences for use with yeast hostsinclude the promoters for 3-phosphoglycerate kinase [Hitzeman et al., J.Biol. Chem., 255:2073 (1980)] or other glycolytic enzymes [Hess et al.,J. Adv. Enzyme Reg., 7:149 (1968); Holland, Biochemistry, 17:4900(1978)], such as enolase, glyceraldehyde-3-phosphate dehydrogenase,hexokinase, pyruvate decarboxylase, phosphofructokinase,glucose-6-phosphate isomerase, 3-phosphoglycerate mutase, pyruvatekinase, triosephosphate isomerase, phosphoglucose isomerase, andglucokinase.

[0123] Other yeast promoters, which are inducible promoters having theadditional advantage of transcription controlled by growth conditions,are the promoter regions for alcohol dehydrogenase 2, isocytochrome C,acid phosphatase, degradative enzymes associated with nitrogenmetabolism, metallothionein, glyceraldehyde-3-phosphate dehydrogenase,and enzymes responsible for maltose and galactose utilization. Suitablevectors and promoters for use in yeast expression are further describedin EP 73,657. Yeast enhancers also are advantageously used with yeastpromoters.

[0124] Apo-2 transcription from vectors in mammalian host cells iscontrolled, for example, by promoters obtained from the genomes ofviruses such as polyoma virus, fowlpox virus (UK 2,211,504 publishedJul. 5, 1989), adenovirus (such as Adenovirus 2), bovine papillomavirus, avian sarcoma virus, cytomegalovirus, a retrovirus, hepatitis-Bvirus and most preferably Simian Virus 40 (SV40), from heterologousmammalian promoters, e.g., the actin promoter or an immunoglobulinpromoter, from heat-shock promoters, and from the promoter normallyassociated with the Apo-2 sequence, provided such promoters arecompatible with the host cell systems.

[0125] The early and late promoters of the SV40 virus are convenientlyobtained as an SV40 restriction fragment that also contains the SV40viral origin of replication [Fiers et al., Nature, 273:113 (1978);Mulligan and Berg, Science, 209:1422-1427 (1980); Pavlakis et al., Proc.Natl. Acad. Sci. USA, 78:7398-7402 (1981)]. The immediate early promoterof the human cytomegalovirus is conveniently obtained as a HindIII Erestriction fragment [Greenaway et al., Gene, 18:355-360 (1982)]. Asystem for expressing DNA in mammalian hosts using the bovine papillomavirus as a vector is disclosed in U.S. Pat. No. 4,419,446. Amodification of this system is described in U.S. Pat. No. 4,601,978 [Seealso Gray et al., Nature, 295:503-508 (1982) on expressing cDNA encodingimmune interferon in monkey cells; Reyes et al., Nature, 297:598-601(1982) on expression of human β-interferon cDNA in mouse cells under thecontrol of a thymidine kinase promoter from herpes simplex virus;Canaani and Berg, Proc. Natl. Acad. Sci. USA 79:5166-5170 (1982) onexpression of the human interferon gene in cultured mouse and rabbitcells; and Gorman et al., Proc. Natl. Acad. Sci. USA, 79:6777-6781(1982) on expression of bacterial CAT sequences in CV-1 monkey kidneycells, chicken embryo fibroblasts, Chinese hamster ovary cells, HeLacells, and mouse NIH-3T3 cells using the Rous sarcoma virus longterminal repeat as a promoter].

[0126] (v) Enhancer Element Component

[0127] Transcription of a DNA encoding the Apo-2 of this invention byhigher eukaryotes may be increased by inserting an enhancer sequenceinto the vector. Enhancers are cis-acting elements of DNA, usually aboutfrom 10 to 300 bp, that act on a promoter to increase its transcription.Enhancers are relatively orientation and position independent, havingbeen found 5′ [Laimins et al., Proc. Natl. Acad. Sci. USA, 78:993(1981]) and 3′ [Lusky et al., Mol. Cell Bio., 3:1108 (1983]) to thetranscription unit, within an intron [Banerji et al., Cell, 33:729(1983)], as well as within the coding sequence itself [Osborne et al.,Mol. Cell Bio., 4:1293 (1984)]. Many enhancer sequences are now knownfrom mammalian genes (globin, elastase, albumin, α-fetoprotein, andinsulin). Typically, however, one will use an enhancer from a eukaryoticcell virus. Examples include the SV40 enhancer on the late side of thereplication origin (bp 100-270), the cytomegalovirus early promoterenhancer, the polyoma enhancer on the late side of the replicationorigin, and adenovirus enhancers. See also Yaniv, Nature, 297:17-18(1982) on enhancing elements for activation of eukaryotic promoters. Theenhancer may be spliced into the vector at a position 5′ or 3′ to theApo-2 coding sequence, but is preferably located at a site 5′ from thepromoter.

[0128] (vi) Transcription Termination Component

[0129] Expression vectors used in eukaryotic host cells (yeast, fungi,insect, plant, animal, human, or nucleated cells from othermulticellular organisms) will also contain sequences necessary for thetermination of transcription and for stabilizing the mRNA. Suchsequences are commonly available from the 5′ and, occasionally 3′,untranslated regions of eukaryotic or viral DNAs or cDNAs. These regionscontain nucleotide segments transcribed as polyadenylated fragments inthe untranslated portion of the mRNA encoding Apo-2.

[0130] (vii) Construction and Analysis of Vectors

[0131] Construction of suitable vectors containing one or more of theabove-listed components employs standard ligation techniques. Isolatedplasmids or DNA fragments are cleaved, tailored, and re-ligated in theform desired to generate the plasmids required.

[0132] For analysis to confirm correct sequences in plasmidsconstructed, the ligation mixtures can be used to transform E. coli K12strain 294 (ATCC 31,446) and successful transformants selected byampicillin or tetracycline resistance where appropriate. Plasmids fromthe transformants are prepared, analyzed by restriction endonucleasedigestion, and/or sequenced by the method of Messing et al., NucleicAcids Res., 9:309 (1981) or by the method of Maxim et al., Methods inEnzymology, 65:499 (1980).

[0133] (viii) Transient Expression Vectors

[0134] Expression vectors that provide for the transient expression inmammalian cells of DNA encoding Apo-2 may be employed. In general,transient expression involves the use of an expression vector that isable to replicate efficiently in a host cell, such that the host cellaccumulates many copies of the expression vector and, in turn,synthesizes high levels of a desired polypeptide encoded by theexpression vector [Sambrook et al., supra]. Transient expressionsystems, comprising a suitable expression vector and a host cell, allowfor the convenient positive identification of polypeptides encoded bycloned DNAs, as well as for the rapid screening of such polypeptides fordesired biological or physiological properties. Thus, transientexpression systems are particularly useful in the invention for purposesof identifying Apo-2 variants.

[0135] (ix) Suitable Exemplary Vertebrate Cell Vectors

[0136] Other methods, vectors, and host cells suitable for adaptation tothe synthesis of Apo-2 in recombinant vertebrate cell culture aredescribed in Gething et al., Nature, 293:620-625 (1981); Mantei et al.,Nature, 281:40-46 (1979); EP 117,060; and EP 117,058.

[0137] 3. Selection and Transformation of Host Cells

[0138] Suitable host cells for cloning or expressing the DNA in thevectors herein are the prokaryote, yeast, or higher eukaryote cellsdescribed above. Suitable prokaryotes for this purpose include but arenot limited to eubacteria, such as Gram-negative or Gram-positiveorganisms, for example, Enterobacteriaceae such as Escherichia, e.g., E.coli, Enterobacter, Erwinia, Klebsiella, Proteus, Salmonella, e.g.,Salmonella typhimurium, Serratia, e.g., Serratia marcescans, andShigella, as well as Bacilli such as B. subtilis and B. licheniformis(e.g., B. licheniformis 41P disclosed in DD 266,710 published Apr. 12,1989), Pseudomonas such as P. aeruginosa, and Streptomyces. Preferably,the host cell should secrete minimal amounts of proteolytic enzymes.

[0139] In addition to prokaryotes, eukaryotic microbes such asfilamentous fungi or yeast are suitable cloning or expression hosts forApo-2-encoding vectors. Saccharomyces cerevisiae, or common baker'syeast, is the most commonly used among lower eukaryotic hostmicroorganisms. However, a number of other genera, species, and strainsare commonly available and useful herein.

[0140] Suitable host cells for the expression of glycosylated Apo-2 arederived from multicellular organisms. Such host cells are capable ofcomplex processing and glycosylation activities. In principle, anyhigher eukaryotic cell culture is workable, whether from vertebrate orinvertebrate culture. Examples of invertebrate cells include plant andinsect cells. Numerous baculoviral strains and variants andcorresponding permissive insect host cells from hosts such as Spodopterafrugiperda (caterpillar), Aedes aegypti (mosquito), Aedes albopictus(mosquito), Drosophila melanogaster (fruitfly), and Bombyx mori havebeen identified [See, e.g., Luckow et al., Bio/Technology, 6:47-55(1988); Miller et al., in Genetic Engineering, Setlow et al., eds., Vol.8 (Plenum Publishing, 1986), pp. 277-279; and Maeda et al., Nature,315:592-594 (1985)]. A variety of viral strains for transfection arepublicly available, e.g., the L-1 variant of Autographa californica NPVand the Bm-5 strain of Bombyx mori NPV.

[0141] Plant cell cultures of cotton, corn, potato, soybean, petunia,tomato, and tobacco can be utilized as hosts. Typically, plant cells aretransfected by incubation with certain strains of the bacteriumAgrobacterium tumefaciens. During incubation of the plant cell culturewith A. tumefaciens, the DNA encoding the Apo-2 can be transferred tothe plant cell host such that it is transfected, and will, underappropriate conditions, express the Apo-2-encoding DNA. In addition,regulatory and signal sequences compatible with plant cells areavailable, such as the nopaline synthase promoter and polyadenylationsignal sequences [Depicker et al., J. Mol. Appl. Gen., 1:561 (1982)]. Inaddition, DNA segments isolated from the upstream region of the T-DNA780 gene are capable of activating or increasing transcription levels ofplant-expressible genes in recombinant DNA-containing plant tissue [EP321,196 published Jun. 21, 1989].

[0142] Propagation of vertebrate cells in culture (tissue culture) isalso well known in the art [See, e.g., Tissue Culture, Academic Press,Kruse and Patterson, editors (1973)]. Examples of useful mammalian hostcell lines are monkey kidney CVI line transformed by SV40 (COS-7, ATCCCRL 1651); human embryonic kidney line (293 or 293 cells subcloned forgrowth in suspension culture, Graham et al., J. Gen Virol., 36:59(1977)); baby hamster kidney cells (BHK, ATCC CCL 10); Chinese hamsterovary cells/-DHFR (CHO, Urlaub and Chasin, Proc. Natl. Acad. Sci. USA,77:4216 (1980)); mouse sertoli cells (TM4, Mather, Biol. Reprod.,23:243-251 (1980)); monkey kidney cells (CV1 ATCC CCL 70); African greenmonkey kidney cells (VERO-76, ATCC CRL-1587); human cervical carcinomacells (HELA, ATCC CCL 2); canine kidney cells (MDCK, ATCC CCL 34);buffalo rat liver cells (BRL 3A, ATCC CRL 1442); human lung cells (W138,ATCC CCL 75); human liver cells (Hep G2, HB 8065); mouse mammary tumor(MMT 060562, ATCC CCL51); TRI cells (Mather et al., Annals N.Y. Acad.Sci., 383:44-68 (1982)); MRC 5 cells; and FS4 cells.

[0143] Host cells are transfected and preferably transformed with theabove-described expression or cloning vectors for Apo-2 production andcultured in conventional nutrient media modified as appropriate forinducing promoters, selecting transformants, or amplifying the genesencoding the desired sequences.

[0144] Transfection refers to the taking up of an expression vector by ahost cell whether or not any coding sequences are in fact expressed.Numerous methods of transfection are known to the ordinarily skilledartisan, for example, CaPO₄ and electroporation. Successful transfectionis generally recognized when any indication of the operation of thisvector occurs within the host cell.

[0145] Transformation means introducing DNA into an organism so that theDNA is replicable, either as an extrachromosomal element or bychromosomal integrant. Depending on the host cell used, transformationis done using standard techniques appropriate to such cells. The calciumtreatment employing calcium chloride, as described in Sambrook et al.,supra, or electroporation is generally used for prokaryotes or othercells that contain substantial cell-wall barriers. Infection withAgrobacterium tumefaciens is used for transformation of certain plantcells, as described by Shaw et al., Gene, 23:315 (1983) and WO 89/05859published Jun. 29, 1989. In addition, plants may be transfected usingultrasound treatment as described in WO 91/00358 published Jan. 10,1991.

[0146] For mammalian cells without such cell walls, the calciumphosphate precipitation method of Graham and van der Eb, Virology,52:456-457 (1978) is preferred. General aspects of mammalian cell hostsystem transformations have been described in U.S. Pat. No. 4,399,216.Transformations into yeast are typically carried out according to themethod of Van Solingen et al., J. Bact., 130:946 (1977) and Hsiao etal., Proc. Natl. Acad. Sci. (USA), 76:3829 (1979). However, othermethods for introducing DNA into cells, such as by nuclearmicroinjection, electroporation, bacterial protoplast fusion with intactcells, or polycations, e.g., polybrene, polyornithine, may also be used.For various techniques for transforming mammalian cells, see Keown etal., Methods in Enzymology, 185:527-537 (1990) and Mansour et al.,Nature, 336:348-352 (1988).

[0147] 4. Culturing the Host Cells

[0148] Prokaryotic cells used to produce Apo-2 may be cultured insuitable media as described generally in Sambrook et al., supra.

[0149] The mammalian host cells used to produce Apo-2 may be cultured ina variety of media. Examples of commercially available media includeHam's F10 (Sigma), Minimal Essential Medium (“MEM”, Sigma), RPMI-1640(Sigma), and Dulbecco's Modified Eagle's Medium (“DMEM”, Sigma). Anysuch media may be supplemented as necessary with hormones and/or othergrowth factors (such as insulin, transferrin, or epidermal growthfactor), salts (such as sodium chloride, calcium, magnesium, andphosphate), buffers (such as HEPES), nucleosides (such as adenosine andthymidine), antibiotics (such as Gentamycin drug), trace elements(defined as inorganic compounds usually present at final concentrationsin the micromolar range), and glucose or an equivalent energy source.Any other necessary supplements may also be included at appropriateconcentrations that would be known to those skilled in the art. Theculture conditions, such as temperature, pH, and the like, are thosepreviously used with the host cell selected for expression, and will beapparent to the ordinarily skilled artisan.

[0150] In general, principles, protocols, and practical techniques formaximizing the productivity of mammalian cell cultures can be found inMammalian Cell Biotechnology: a Practical Approach, M. Butler, ed. (IRLPress, 1991).

[0151] The host cells referred to in this disclosure encompass cells inculture as well as cells that are within a host animal.

[0152] 5. Detecting Gene Amplification/Expression

[0153] Gene amplification and/or expression may be measured in a sampledirectly, for example, by conventional Southern blotting, Northernblotting to quantitate the transcription of mRNA [Thomas, Proc. Natl.Acad. Sci. USA, 77:5201-5205 (1980)], dot blotting (DNA analysis), or insitu hybridization, using an appropriately labeled probe, based on thesequences provided herein. Various labels may be employed, most commonlyradioisotopes, and particularly ³²P. However, other techniques may alsobe employed, such as using biotin-modified nucleotides for introductioninto a polynucleotide. The biotin then serves as the site for binding toavidin or antibodies, which may be labeled with a wide variety oflabels, such as radionucleotides, fluorescers or enzymes. Alternatively,antibodies may be employed that can recognize specific duplexes,including DNA duplexes, RNA duplexes, and DNA-RNA hybrid duplexes orDNA-protein duplexes. The antibodies in turn may be labeled and theassay may be carried out where the duplex is bound to a surface, so thatupon the formation of duplex on the surface, the presence of antibodybound to the duplex can be detected.

[0154] Gene expression, alternatively, may be measured by immunologicalmethods, such as immunohistochemical staining of cells or tissuesections and assay of cell culture or body fluids, to quantitatedirectly the expression of gene product. With immunohistochemicalstaining techniques, a cell sample is prepared, typically by dehydrationand fixation, followed by reaction with labeled antibodies specific forthe gene product coupled, where the labels are usually visuallydetectable, such as enzymatic labels, fluorescent labels, or luminescentlabels.

[0155] Antibodies useful for immunohistochemical staining and/or assayof sample fluids may be either monoclonal or polyclonal, and may beprepared in any mammal. Conveniently, the antibodies may be preparedagainst a native sequence Apo-2 polypeptide or against a syntheticpeptide based on the DNA sequences provided herein or against exogenoussequence fused to Apo-2 DNA and encoding a specific antibody epitope.

[0156] 6. Purification of Apo-2 Polypeptide

[0157] Forms of Apo-2 may be recovered from culture medium or from hostcell lysates. If the Apo-2 is membrane-bound, it can be released fromthe membrane using a suitable detergent solution (e.g. Triton-X 100) orits extracellular domain may be released by enzymatic cleavage.

[0158] When Apo-2 is produced in a recombinant cell other than one ofhuman origin, the Apo-2 is free of proteins or polypeptides of humanorigin. However, it may be desired to purify Apo-2 from recombinant cellproteins or polypeptides to obtain preparations that are substantiallyhomogeneous as to Apo-2. As a first step, the culture medium or lysatemay be centrifuged to remove particulate cell debris. Apo-2 thereafteris purified from contaminant soluble proteins and polypeptides, with thefollowing procedures being exemplary of suitable purificationprocedures: by fractionation on an ion-exchange column; ethanolprecipitation; reverse phase HPLC; chromatography on silica or on acation-exchange resin such as DEAE; chromatofocusing; SDS-PAGE; ammoniumsulfate precipitation; gel filtration using, for example, Sephadex G-75;and protein A Sepharose columns to remove contaminants such as IgG.

[0159] Apo-2 variants in which residues have been deleted, inserted, orsubstituted can be recovered in the same fashion as native sequenceApo-2, taking account of changes in properties occasioned by thevariation. For example, preparation of an Apo-2 fusion with anotherprotein or polypeptide, e.g., a bacterial or viral antigen,immunoglobulin sequence, or receptor sequence, may facilitatepurification; an immunoaffinity column containing antibody to thesequence can be used to adsorb the fusion polypeptide. Other types ofaffinity matrices also can be used.

[0160] A protease inhibitor such as phenyl methyl sulfonyl fluoride(PMSF) also may be useful to inhibit proteolytic degradation duringpurification, and antibiotics may be included to prevent the growth ofadventitious contaminants. One skilled in the art will appreciate thatpurification methods suitable for native sequence Apo-2 may requiremodification to account for changes in the character of Apo-2 or itsvariants upon expression in recombinant cell culture.

[0161] 7. Covalent Modifications of Apo-2 Polypeptides

[0162] Covalent modifications of Apo-2 are included within the scope ofthis invention. One type of covalent modification of the Apo-2 isintroduced into the molecule by reacting targeted amino acid residues ofthe Apo-2 with an organic derivatizing agent that is capable of reactingwith selected side chains or the N- or C-terminal residues of the Apo-2.

[0163] Derivatization with bifunctional agents is useful forcrosslinking Apo-2 to a water-insoluble support matrix or surface foruse in the method for purifying anti-Apo-2 antibodies, and vice-versa.Derivatization with one or more bifunctional agents will also be usefulfor crosslinking Apo-2 molecules to generate Apo-2 dimers. Such dimersmay increase binding avidity and extend half-life of the molecule invivo. Commonly used crosslinking agents include, e.g.,1,1-bis(diazoacetyl)-2-phenylethane, glutaraldehyde,N-hydroxysuccinimide esters, for example, esters with 4-azidosalicylicacid, homobifunctional imidoesters, including disuccinimidyl esters suchas 3,3′-dithiobis(succinimidyl-propionate), and bifunctional maleimidessuch as bis-N-maleimido-1,8-octane. Derivatizing agents such asmethyl-3-[(p-azidophenyl)-dithio]propioimidate yield photoactivatableintermediates that are capable of forming crosslinks in the presence oflight. Alternatively, reactive water-insoluble matrices such as cyanogenbromide-activated carbohydrates and the reactive substrates described inU.S. Pat. Nos. 3,969,287; 3,691,016; 4,195,128; 4,247,642; 4,229,537;and 4,330,440 are employed for protein immobilization.

[0164] Other modifications include deamidation of glutaminyl andasparaginyl residues to the corresponding glutamyl and aspartylresidues, respectively, hydroxylation of proline and lysine,phosphorylation of hydroxyl groups of seryl or threonyl residues,methylation of the α-amino groups of lysine, arginine, and histidineside chains [T. E. Creighton, Proteins: Structure and MolecularProperties, W. H. Freeman & Co., San Francisco, pp. 79-86 (1983)],acetylation of the N-terminal amine, and amidation of any C-terminalcarboxyl group. The modified forms of the residues fall within the scopeof the present invention.

[0165] Another type of covalent modification of the Apo-2 polypeptideincluded within the scope of this invention comprises altering thenative glycosylation pattern of the polypeptide. “Altering the nativeglycosylation pattern” is intended for purposes herein to mean deletingone or more carbohydrate moieties found in native sequence Apo-2, and/oradding one or more glycosylation sites that are not present in thenative sequence Apo-2.

[0166] Glycosylation of polypeptides is typically either N-linked orO-linked. N-linked refers to the attachment of the carbohydrate moietyto the side chain of an asparagine residue. The tripeptide sequencesasparagine-X-serine and asparagine-X-threonine, where X is any aminoacid except proline, are the recognition sequences for enzymaticattachment of the carbohydrate moiety to the asparagine side chain.Thus, the presence of either of these tripeptide sequences in apolypeptide creates a potential glycosylation site. O-linkedglycosylation refers to the attachment of one of the sugarsN-aceylgalactosamine, galactose, or xylose to a hydroxylamino acid, mostcommonly serine or threonine, although 5-hydroxyproline or5-hydroxylysine may also be used.

[0167] Addition of glycosylation sites to the Apo-2 polypeptide may beaccomplished by altering the amino acid sequence such that it containsone or more of the above-described tripeptide sequences (for N-linkedglycosylation sites). The alteration may also be made by the additionof, or substitution by, one or more serine or threonine residues to thenative sequence Apo-2 (for O-linked glycosylation sites). The Apo-2amino acid sequence may optionally be altered through changes at the DNAlevel, particularly by mutating the DNA encoding the Apo-2 polypeptideat preselected bases such that codons are generated that will translateinto the desired amino acids. The DNA mutation(s) may be made usingmethods described above and in U.S. Pat. No. 5,364,934, supra.

[0168] Another means of increasing the number of carbohydrate moietieson the Apo-2 polypeptide is by chemical or enzymatic coupling ofglycosides to the polypeptide. Depending on the coupling mode used, thesugar(s) may be attached to (a) arginine and histidine, (b) freecarboxyl groups, (c) free sulfhydryl groups such as those of cysteine,(d) free hydroxyl groups such as those of serine, threonine, orhydroxyproline, (e) aromatic residues such as those of phenylalanine,tyrosine, or tryptophan, or (f) the amide group of glutamine. Thesemethods are described in WO 87/05330 published Sep. 11, 1987, and inAplin and Wriston, CRC Crit. Rev. Biochem., pp. 259-306 (1981).

[0169] Removal of carbohydrate moieties present on the Apo-2 polypeptidemay be accomplished chemically or enzymatically or by mutationalsubstitution of codons encoding for amino acid residues that serve astargets for glycosylation. For instance, chemical deglycosylation byexposing the polypeptide to the compound trifluoromethanesulfonic acid,or an equivalent compound can result in the cleavage of most or allsugars except the linking sugar (N-acetylglucosamine orN-acetylgalactosamine), while leaving the polypeptide intact. Chemicaldeglycosylation is described by Hakimuddin, et al., Arch. Biochem.Biophys., 259:52 (1987) and by Edge et al., Anal. Biochem., 118:131(1981). Enzymatic cleavage of carbohydrate moieties on polypeptides canbe achieved by the use of a variety of endo- and exo-glycosidases asdescribed by Thotakura et al., Meth. Enzymol., 138:350 (1987).

[0170] Glycosylation at potential glycosylation sites may be preventedby the use of the compound tunicamycin as described by Duksin et al., J.Biol. Chem., 257:3105 (1982). Tunicamycin blocks the formation ofprotein-N-glycoside linkages.

[0171] Another type of covalent modification of Apo-2 comprises linkingthe Apo-2 polypeptide to one of a variety of nonproteinaceous polymers,e.g., polyethylene glycol, polypropylene glycol, or polyoxyalkylenes, inthe manner set forth in U.S. Pat. Nos. 4,640,835; 4,496,689; 4,301,144;4,670,417; 4,791,192 or 4,179,337.

[0172] 8. Apo-2 Chimeras

[0173] The present invention also provides chimeric molecules comprisingApo-2 fused to another, heterologous polypeptide or amino acid sequence.

[0174] In one embodiment, the chimeric molecule comprises a fusion ofthe Apo-2 with a tag polypeptide which provides an epitope to which ananti-tag antibody can selectively bind. The epitope tag is generallyplaced at the amino- or carboxyl-terminus of the Apo-2. The presence ofsuch epitope-tagged forms of the Apo-2 can be detected using an antibodyagainst the tag polypeptide. Also, provision of the epitope tag enablesthe Apo-2 to be readily purified by affinity purification using ananti-tag antibody or another type of affinity matrix that binds to theepitope tag.

[0175] Various tag polypeptides and their respective antibodies are wellknown in the art. Examples include the flu HA tag polypeptide and itsantibody 12CA5 [Field et al., Mol. Cell. Biol., 8:2159-2165 (1988)]; thec-myc tag and the 8F9, 3C7, 6E10, G4, B7 and 9E10 antibodies thereto[Evan et al., Molecular and Cellular Biology, 5:3610-3616 (1985)]; andthe Herpes Simplex virus glycoprotein D (gD) tag and its antibody[Paborsky et al., Protein Engineering, 3 (6):547-553 (1990)1. Other tagpolypeptides include the Flag-peptide [Hopp et al., BioTechnology,6:1204-1210 (1988)]; the KT3 epitope peptide [Martin et al., Science,255:192-194 (1992)]; an α-tubulin epitope peptide [Skinner et al., J.Biol. Chem., 266:15163-15166 (1991)]; and the T7 gene 10 protein peptidetag [Lutz-Freyermuth et al., Proc. Natl. Acad. Sci. USA, 87:6393-6397(1990)]. Once the tag polypeptide has been selected, an antibody theretocan be generated using the techniques disclosed herein.

[0176] Generally, epitope-tagged Apo-2 may be constructed and producedaccording to the methods described above. Epitope-tagged Apo-2 is alsodescribed in the Examples below. Apo-2-tag polypeptide fusions arepreferably constructed by fusing the cDNA sequence encoding the Apo-2portion in-frame to the tag polypeptide DNA sequence and expressing theresultant DNA fusion construct in appropriate host cells. Ordinarily,when preparing the Apo-2-tag polypeptide chimeras of the presentinvention, nucleic acid encoding the Apo-2 will be fused at its 3′ endto nucleic acid encoding the N-terminus of the tag polypeptide, however5′ fusions are also possible. For example, a polyhistidine sequence ofabout 5 to about 10 histidine residues may be fused at the N-terminus orthe C-terminus and used as a purification handle in affinitychromatography.

[0177] Epitope-tagged Apo-2 can be purified by affinity chromatographyusing the anti-tag antibody. The matrix to which the affinity antibodyis attached may include, for instance, agarose, controlled pore glass orpoly(styrenedivinyl)benzene. The epitope-tagged Apo-2 can then be elutedfrom the affinity column using techniques known in the art.

[0178] In another embodiment, the chimeric molecule comprises an Apo-2polypeptide fused to an immunoglobulin sequence. The chimeric moleculemay also comprise a particular domain sequence of Apo-2, such as anextracellular domain sequence of Apo-2 fused to an immunoglobulinsequence. This includes chimeras in monomeric, homo- orheteromultimeric, and particularly homo- or heterodimeric, or-tetrameric forms; optionally, the chimeras may be in dimeric forms orhomodimeric heavy chain forms. Generally, these assembledimmunoglobulins will have known unit structures as represented by thefollowing diagrams.

[0179] A basic four chain structural unit is the form in which IgG, IgD,and IgE exist. A four chain unit is repeated in the higher molecularweight immunoglobulins; IgM generally exists as a pentamer of basicfour-chain units held together by disulfide bonds. IgA globulin, andoccasionally IgG globulin, may also exist in a multimeric form in serum.In the case of multimers, each four chain unit may be the same ordifferent.

[0180] The following diagrams depict some exemplary monomer, homo- andheterodimer and homo- and heteromultimer structures. These diagrams aremerely illustrative, and the chains of the multimers are believed to bedisulfide bonded in the same fashion as native immunoglobulins.

[0181] In the foregoing diagrams, “A” means an Apo-2 sequence or anApo-2 sequence fused to a heterologous sequence; X is an additionalagent, which may be the same as A or different, a portion of animmunoglobulin superfamily member such as a variable region or avariable region-like domain, including a native or chimericimmunoglobulin variable region, a toxin such a pseudomonas exotoxin orricin, or a sequence functionally binding to another protein, such asother cytokines (i.e., IL-1, interferon-γ) or cell surface molecules(i.e., NGFR, CD40, OX40, Fas antigen, T2 proteins of Shope and myxomapoxviruses), or a polypeptide therapeutic agent not otherwise normallyassociated with a constant domain; Y is a linker or another receptorsequence; and V_(L), V_(H), C_(L) and C_(H) represent light or heavychain variable or constant domains of an immunoglobulin. Structurescomprising at least one CRD of an Apo-2 sequence as “A” and anothercell-surface protein having a repetitive pattern of CRDs (such as TNFR)as “X” are specifically included.

[0182] It will be understood that the above diagrams are merelyexemplary of the possible structures of the chimeras of the presentinvention, and do not encompass all possibilities. For example, theremight desirably be several different “A”s, “X”s, or “Y”s in any of theseconstructs. Also, the heavy or light chain constant domains may beoriginated from the same or different immunoglobulins. All possiblepermutations of the illustrated and similar structures are all withinthe scope of the invention herein.

[0183] In general, the chimeric molecules can be constructed in afashion similar to chimeric antibodies in which a variable domain froman antibody of one species is substituted for the variable domain ofanother species. See, for example, EP 0 125 023; EP 173,494; Munro,Nature, 312:597 (Dec. 13, 1984); Neuberger et al., Nature, 312:604-608(Dec. 13, 1984); Sharon et al., Nature, 309:364-367 (May 24, 1984);Morrison et al., Proc. Nat'l. Acad. Sci. USA, 81:6851-6855 (1984);Morrison et al., Science, 229:1202-1207 (1985); Boulianne et al.,Nature, 312:643-646 (Dec. 13, 1984); Capon et al., Nature, 337:525-531(1989); Traunecker et al., Nature, 339:68-70 (1989).

[0184] Alternatively, the chimeric molecules may be constructed asfollows. The DNA including a region encoding the desired sequence, suchas an Apo-2 and/or TNFR sequence, is cleaved by a restriction enzyme ator proximal to the 3′ end of the DNA encoding the immunoglobulin-likedomain(s) and at a point at or near the DNA encoding the N-terminal endof the Apo-2 or TNFR polypeptide (where use of a different leader iscontemplated) or at or proximal to the N-terminal coding region for TNFR(where the native signal is employed). This DNA fragment then is readilyinserted proximal to DNA encoding an immunoglobulin light or heavy chainconstant region and, if necessary, the resulting construct tailored bydeletional mutagenesis. Preferably, the Ig is a human immunoglobulinwhen the chimeric molecule is intended for in vivo therapy for humans.DNA encoding immunoglobulin light or heavy chain constant regions isknown or readily available from cDNA Libraries or is synthesized. Seefor example, Adams et al., Biochemistry, 19:2711-2719 (1980); Gough etal., Biochemistry, 19:2702-2710 (1980); Dolby et al., Proc. Natl. Acad.Sci. USA, 77:6027-6031 (1980); Rice et al., Proc. Natl. Acad. Sci.,79:7862-7865 (1982); Falkner et al., Nature, 298:286-288 (1982); andMorrison et al., Ann. Rev. Immunol., 2:239-256 (1984).

[0185] Further details of how to prepare such fusions are found inpublications concerning the preparation of immunoadhesins.Immunoadhesins in general, and CD4-Ig fusion molecules specifically aredisclosed in WO 89/02922, published Apr. 6, 1989. Molecules comprisingthe extracellular portion of CD4, the receptor for humanimmunodeficiency virus (HIV), linked to IgG heavy chain constant regionare known in the art and have been found to have a markedly longerhalf-life and lower clearance than the soluble extracellular portion ofCD4 [Capon et al., supra; Byrn et al., Nature, 344:667 (1990)]. Theconstruction of specific chimeric TNFR-IgG molecules is also describedin Ashkenazi et al. Proc. Natl. Acad. Sci., 88:10535-10539 (1991);Lesslauer et al. [J. Cell. Biochem. Supplement 15F, 1991, p. 115 (P432)]; and Peppel and Beutler, J. Cell. Biochem. Supplement 15F, 1991,p. 118 (P 439)].

[0186] B. Therapeutic and Non-therapeutic Uses for Apo-2

[0187] Apo-2, as disclosed in the present specification, can be employedtherapeutically to induce apoptosis in mammalian cells. This therapy canbe accomplished for instance, using in vivo or ex vivo gene therapytechniques and includes the use of the death domain sequences disclosedherein. The Apo-2 chimeric molecules (including the chimeric moleculescontaining an extracellular domain sequence of Apo-2) comprisingimmunoglobulin sequences can also be employed therapeutically to inhibitapoptosis or NF-KB induction by Apo-2L or by another ligand that Apo-2binds to.

[0188] The Apo-2 of the invention also has utility in non-therapeuticapplications. Nucleic acid sequences encoding the Apo-2 may be used as adiagnostic for tissue-specific typing. For example, procedures like insitu hybridization, Northern and Southern blotting, and PCR analysis maybe used to determine whether DNA and/or RNA encoding Apo-2 is present inthe cell type(s) being evaluated. Apo-2 nucleic acid will also be usefulfor the preparation of Apo-2 by the recombinant techniques describedherein.

[0189] The isolated Apo-2 may be used in quantitative diagnostic assaysas a control against which samples containing unknown quantities ofApo-2 may be prepared. Apo-2 preparations are also useful in generatingantibodies, as standards in assays for Apo-2 (e.g., by labeling Apo-2for use as a standard in a radioimmunoassay, radioreceptor assay, orenzyme-linked immunoassay), in affinity purification techniques, and incompetitive-type receptor binding assays when labeled with, forinstance, radioiodine, enzymes, or fluorophores.

[0190] Modified forms of the Apo-2, such as the Apo-2-IgG chimericmolecules (immunoadhesins) described above, can be used as immunogens inproducing anti-Apo-2 antibodies.

[0191] Nucleic acids which encode Apo-2 or its modified forms can alsobe used to generate either transgenic animals or “knock out” animalswhich, in turn, are useful in the development and screening oftherapeutically useful reagents. A transgenic animal (e.g., a mouse orrat) is an animal having cells that contain a transgene, which transgenewas introduced into the animal or an ancestor of the animal at aprenatal, e.g., an embryonic stage. A transgene is a DNA which isintegrated into the genome of a cell from which a transgenic animaldevelops. In one embodiment, cDNA encoding Apo-2 or an appropriatesequence thereof (such as Apo-2-IgG) can be used to clone genomic DNAencoding Apo-2 in accordance with established techniques and the genomicsequences used to generate transgenic animals that contain cells whichexpress DNA encoding Apo-2. Methods for generating transgenic animals,particularly animals such as mice or rats, have become conventional inthe art and are described, for example, in U.S. Pat. Nos. 4,736,866 and4,870,009. Typically, particular cells would be targeted for Apo-2transgene incorporation with tissue-specific enhancers. Transgenicanimals that include a copy of a transgene encoding Apo-2 introducedinto the germ line of the animal at an embryonic stage can be used toexamine the effect of increased expression of DNA encoding Apo-2. Suchanimals can be used as tester animals for reagents thought to conferprotection from, for example, pathological conditions associated withexcessive apoptosis. In accordance with this facet of the invention, ananimal is treated with the reagent and a reduced incidence of thepathological condition, compared to untreated animals bearing thetransgene, would indicate a potential therapeutic intervention for thepathological condition. In another embodiment, transgenic animals thatcarry a soluble form of Apo-2 such as an Apo-2 ECD or an immunoglobulinchimera of such form could be constructed to test the effect of chronicneutralization of Apo-2L, a ligand of Apo-2.

[0192] Alternatively, non-human homologues of Apo-2 can be used toconstruct an Apo-2 “knock out” animal which has a defective or alteredgene encoding Apo-2 as a result of homologous recombination between theendogenous gene encoding Apo-2 and altered genomic DNA encoding Apo-2introduced into an embryonic cell of the animal. For example, cDNAencoding Apo-2 can be used to clone genomic DNA encoding Apo-2 inaccordance with established techniques. A portion of the genomic DNAencoding Apo-2 can be deleted or replaced with another gene, such as agene encoding a selectable marker which can be used to monitorintegration. Typically, several kilobases of unaltered flanking DNA(both at the 5′ and 3′ ends) are included in the vector [see e.g.,Thomas and Capecchi, Cell, 51:503 (1987) for a description of homologousrecombination vectors]. The vector is introduced into an embryonic stemcell line (e.g., by electroporation) and cells in which the introducedDNA has homologously recombined with the endogenous DNA are selected[see e.g., Li et al., Cell, 69:915 (1992)]. The selected cells are theninjected into a blastocyst of an animal (e.g., a mouse or rat) to formaggregation chimeras [see e.g., Bradley, in Teratocarcinomas andEmbryonic Stem Cells: A Practical Approach, E. J. Robertson, ed. (IRL,Oxford, 1987), pp. 113-152]. A chimeric embryo can then be implantedinto a suitable pseudopregnant female foster animal and the embryobrought to term to create a “knock out” animal. Progeny harboring thehomologously recombined DNA in their germ cells can be identified bystandard techniques and used to breed animals in which all cells of theanimal contain the homologously recombined DNA. Knockout animals can becharacterized for instance, for their ability to defend against certainpathological conditions and for their development of pathologicalconditions due to absence of the Apo-2 polypeptide, including forexample, development of tumors.

[0193] C. Anti-Apo-2 Antibody Preparation

[0194] The present invention further provides anti-Apo-2 antibodies.Antibodies against Apo-2 may be prepared as follows. Exemplaryantibodies include polyclonal, monoclonal, humanized, bispecific, andheteroconjugate antibodies.

[0195] 1. Polyclonal Antibodies

[0196] The Apo-2 antibodies may comprise polyclonal antibodies. Methodsof preparing polyclonal antibodies are known to the skilled artisan.Polyclonal antibodies can be raised in a mammal, for example, by one ormore injections of an immunizing agent and, if desired, an adjuvant.Typically, the immunizing agent and/or adjuvant will be injected in themammal by multiple subcutaneous or intraperitoneal injections. Theimmunizing agent may include the Apo-2 polypeptide or a fusion proteinthereof. An example of a suitable immunizing agent is an Apo-2-IgGfusion protein, such as an Apo-2 ECD-IgG fusion protein. Cellsexpressing Apo-2 at their surface may also be employed. It may be usefulto conjugate the immunizing agent to a protein known to be immunogenicin the mammal being immunized. Examples of such immunogenic proteinswhich may be employed include but are not limited to keyhole limpethemocyanin, serum albumin, bovine thyroglobulin, and soybean trypsininhibitor. An aggregating agent such as alum may also be employed toenhance the mammal's immune response. Examples of adjuvants which may beemployed include Freund's complete adjuvant and MPL-TDM adjuvant(monophosphoryl Lipid A, synthetic trehalose dicorynomycolate). Theimmunization protocol may be selected by one skilled in the art withoutundue experimentation. The mammal can then be bled, and the serumassayed for antibody titer. If desired, the mammal can be boosted untilthe antibody titer increases or plateaus.

[0197] 2. Monoclonal Antibodies

[0198] The Apo-2 antibodies may, alternatively, be monoclonalantibodies. Monoclonal antibodies may be prepared using hybridomamethods, such as those described by Kohler and Milstein, supra. In ahybridoma method, a mouse, hamster, or other appropriate host animal, istypically immunized (such as described above) with an immunizing agentto elicit lymphocytes that produce or are capable of producingantibodies that will specifically bind to the immunizing agent.Alternatively, the lymphocytes may be immunized in vitro.

[0199] The immunizing agent will typically include the Apo-2 polypeptideor a fusion protein thereof. An example of a suitable immunizing agentis an Apo-2-IgG fusion protein or chimeric molecule. A specific exampleof an Apo-2 ECD-IgG immunogen is described in Example 9 below. Cellsexpressing Apo-2 at their surface may also be employed. Generally,either peripheral blood lymphocytes (“PBLs”) are used if cells of humanorigin are desired, or spleen cells or lymph node cells are used ifnon-human mammalian sources are desired. The lymphocytes are then fusedwith an immortalized cell line using a suitable fusing agent, such aspolyethylene glycol, to form a hybridoma cell [Goding, MonoclonalAntibodies: Principles and Practice, Academic Press, (1986) pp. 59-103].Immortalized cell lines are usually transformed mammalian cells,particularly myeloma cells of rodent, bovine and human origin. Usually,rat or mouse myeloma cell lines are employed. The hybridoma cells may becultured in a suitable culture medium that preferably contains one ormore substances that inhibit the growth or survival of the unfused,immortalized cells. For example, if the parental transformed cells lackthe enzyme hypoxanthine guanine phosphoribosyl transferase (HGPRT orHPRT), the culture medium for the hybridomas typically will includehypoxanthine, aminopterin, and thymidine (“HAT medium”), whichsubstances prevent the growth of HGPRT-deficient cells.

[0200] Preferred immortalized cell lines are those that fuseefficiently, support stable high level expression of antibody by theselected antibody-producing cells, and are sensitive to a medium such asHAT medium. More preferred immortalized cell lines are murine myelomalines, which can be obtained, for instance, from the Salk Institute CellDistribution Center, San Diego, Calif. and the American Type CultureCollection, Manassas, Va. Human myeloma and mouse-human heteromyelomacell lines also have been described for the production of humanmonoclonal antibodies [Kozbor, J. Immunol., 133:3001 (1984); Brodeur etal., Monoclonal Antibody Production Techniques and Applications, MarcelDekker, Inc., New York, (1987) pp. 51-63].

[0201] The culture medium in which the hybridoma cells are cultured canthen be assayed for the presence of monoclonal antibodies directedagainst Apo-2. Preferably, the binding specificity of monoclonalantibodies produced by the hybridoma cells is determined byimmunoprecipitation or by an in vitro binding assay, such asradioimmunoassay (RIA) or enzyme-linked immunoabsorbent assay (ELISA).Such techniques and assays are known in the art. The binding affinity ofthe monoclonal antibody can, for example, be determined by the Scatchardanalysis of Munson and Pollard, Anal. Biochem., 107:220 (1980).

[0202] After the desired hybridoma cells are identified, the clones maybe subcloned by limiting dilution procedures and grown by standardmethods [Goding, supra]. Suitable culture media for this purposeinclude, for example, Dulbecco's Modified Eagle's Medium and RPMI-1640medium. Alternatively, the hybridoma cells may be grown in vivo asascites in a mammal.

[0203] The monoclonal antibodies secreted by the subclones may beisolated or purified from the culture medium or ascites fluid byconventional immunoglobulin purification procedures such as, forexample, protein A-Sepharose, hydroxylapatite chromatography, gelelectrophoresis, dialysis, or affinity chromatography.

[0204] The monoclonal antibodies may also be made by recombinant DNAmethods, such as those described in U.S. Pat. No. 4,816,567. DNAencoding the monoclonal antibodies of the invention can be readilyisolated and sequenced using conventional procedures (e.g., by usingoligonucleotide probes that are capable of binding specifically to genesencoding the heavy and light chains of murine antibodies). The hybridomacells of the invention serve as a preferred source of such DNA. Onceisolated, the DNA may be placed into expression vectors, which are thentransfected into host cells such as simian COS cells, Chinese hamsterovary (CHO) cells, or myeloma cells that do not otherwise produceimmunoglobulin protein, to obtain the synthesis of monoclonal antibodiesin the recombinant host cells. The DNA also may be modified, forexample, by substituting the coding sequence for human heavy and lightchain constant domains in place of the homologous murine sequences [U.S.Pat. No. 4,816,567; Morrison et al., supra] or by covalently joining tothe immunoglobulin coding sequence all or part of the coding sequencefor a non-immunoglobulin polypeptide. Such a non-immunoglobulinpolypeptide can be substituted for the constant domains of an antibodyof the invention, or can be substituted for the variable domains of oneantigen-combining site of an antibody of the invention to create achimeric bivalent antibody.

[0205] As described in the Examples below, anti-Apo-2 monoclonalantibodies have been prepared. One of these antibodies, 3F11.39.7, hasbeen deposited with ATCC and has been assigned deposit accession no.HB-12456. In one embodiment, the monoclonal antibodies of the inventionwill have the same biological characteristics as the monoclonalantibodies secreted by the hybridoma cell line(s) deposited underAccession No. HB-12456. The term “biological characteristics” is used torefer to the in vitro and/or in vivo activities or properties of themonoclonal antibody, such as the ability to specifically bind to Apo-2or to substantially block, induce or enhance Apo-2 activation. Asdisclosed in the present specification, the 3F11.39.7 monoclonalantibody (HB-12456) is characterized as having agonistic activity forinducing apoptosis, binding to the Apo-2 receptor, having blockingactivity as described in the Examples below, and having somecross-reactivity to DR4 but not to DcR1 or DcR2. Optionally, themonoclonal antibody will bind to the same epitope as the 3F11.39.7antibody disclosed herein. This can be determined by conducting variousassays, such as described herein and in the Examples. For instance, todetermine whether a monoclonal antibody has the same specificity as the3F11.39.7 antibody specifically disclosed, one can-compare activity inApo-2 blocking and apoptosis induction assays, such as those describedin the Examples below.

[0206] The antibodies of the invention may also comprise monovalentantibodies. Methods for preparing monovalent antibodies are well knownin the art. For example, one method involves recombinant expression ofimmunoglobulin light chain and modified heavy chain. The heavy chain istruncated generally at any point in the Fc region so as to prevent heavychain crosslinking. Alternatively, the relevant cysteine residues aresubstituted with another amino acid residue or are deleted so as toprevent crosslinking.

[0207] In vitro methods are also suitable for preparing monovalentantibodies. Digestion of antibodies to produce fragments thereof,particularly, Fab fragments, can be accomplished using routinetechniques known in the art. For instance, digestion can be performedusing papain. Examples of papain digestion are described in WO 94/29348published Dec. 22, 1994 and U.S. Pat. No. 4,342,566. Papain digestion ofantibodies typically produces two identical antigen binding fragments,called Fab fragments, each with a single antigen binding site, and aresidual Fc fragment. Pepsin treatment yields an F(ab′)₂ fragment thathas two antigen combining sites and is still capable of cross-linkingantigen.

[0208] The Fab fragments produced in the antibody digestion also containthe constant domains of the light chain and the first constant domain(CH₁) of the heavy chain. Fab′ fragments differ from Fab fragments bythe addition of a few residues at the carboxy terminus of the heavychain CH₁ domain including one or more cysteines from the antibody hingeregion. Fab′-SH is the designation herein for Fab′ in which the cysteineresidue(s) of the constant domains bear a free thiol group. F(ab′)₂antibody fragments originally were produced as pairs of Fab′ fragmentswhich have hinge cysteines between them. Other chemical couplings ofantibody fragments are also known.

[0209] 3. Humanized Antibodies

[0210] The Apo-2 antibodies of the invention may further comprisehumanized antibodies or human antibodies. Humanized forms of non-human(e.g., murine) antibodies are chimeric immunoglobulins, immunoglobulinchains or fragments thereof (such as Fv, Fab, Fab′, F(ab′)₂ or otherantigen-binding subsequences of antibodies) which contain minimalsequence derived from non-human immunoglobulin. Humanized antibodiesinclude human immunoglobulins (recipient antibody) in which residuesfrom a complementary determining region (CDR) of the recipient arereplaced by residues from a CDR of a non-human species (donor antibody)such as mouse, rat or rabbit having the desired specificity, affinityand capacity. In some instances, Fv framework residues of the humanimmunoglobulin are replaced by corresponding non-human residues.Humanized antibodies may also comprise residues which are found neitherin the recipient antibody nor in the imported CDR or frameworksequences. In general, the humanized antibody will comprisesubstantially all of at least one, and typically two, variable domains,in which all or substantially all of the CDR regions correspond to thoseof a non-human immunoglobulin and all or substantially all of the FRregions are those of a human immunoglobulin consensus sequence. Thehumanized antibody optimally also will comprise at least a portion of animmunoglobulin constant region (Fc), typically that of a humanimmunoglobulin [Jones et al., Nature, 321:522-525 (1986); Riechmann etal., Nature, 332:323-329 (1988); and Presta, Curr. Op. Struct. Biol.,2:593-596 (1992)].

[0211] Methods for humanizing non-human antibodies are well known in theart. Generally, a humanized antibody has one or more amino acid residuesintroduced into it from a source which is non-human. These non-humanamino-acid residues are often referred to as “import” residues, whichare typically taken from an “import” variable domain. Humanization canbe essentially performed following the method of Winter and co-workers[Jones et al., Nature, 321:522-525 (1986); Riechmann et al., Nature,332:323-327 (1988); Verhoeyen et al., Science, 239:1534-1536 (1988)], bysubstituting rodent CDRs or CDR sequences for the correspondingsequences of a human antibody. Accordingly, such “humanized” antibodiesare chimeric antibodies (U.S. Pat. No. 4,816,567), wherein substantiallyless than an intact human variable domain has been substituted by thecorresponding sequence from a non-human species. In practice, humanizedantibodies are typically human antibodies in which some CDR residues andpossibly some FR residues are substituted by residues from analogoussites in rodent antibodies.

[0212] The choice of human variable domains, both light and heavy, to beused in making the humanized antibodies is very important in order toreduce antigenicity. According to the “best-fit” method, the sequence ofthe variable domain of a rodent antibody is screened against the entirelibrary of known human variable domain sequences. The human sequencewhich is closest to that of the rodent is then accepted as the humanframework (FR) for the humanized antibody [Sims et al., J. Immunol.,151:2296 (1993); Chothia and Lesk, J. Mol. Biol., 196:901 (1987)].Another method uses a particular framework derived from the consensussequence of all human antibodies of a particular subgroup of light orheavy chains. The same framework may be used for several differenthumanized antibodies [Carter et al., Proc. Natl. Acad. Sci. USA, 89:4285(1992); Presta et al., J. Immunol., 151:2623 (1993)].

[0213] It is further important that antibodies be humanized withretention of high affinity for the antigen and other favorablebiological properties. To achieve this goal, according to a preferredmethod, humanized antibodies are prepared by a process of analysis ofthe parental sequences and various conceptual humanized products usingthree dimensional models of the parental and humanized sequences. Threedimensional immunoglobulin models are commonly available and arefamiliar to those skilled in the art. Computer programs are availablewhich illustrate and display probable three-dimensional conformationalstructures of selected candidate immunoglobulin sequences. Inspection ofthese displays permits analysis of the likely role of the residues inthe functioning of the candidate immunoglobulin sequence, i.e., theanalysis of residues that influence the ability of the candidateimmunoglobulin to bind its antigen. In this way, FR residues can beselected and combined from the consensus and import sequence so that thedesired antibody characteristic, such as increased affinity for thetarget antigen(s), is achieved. In general, the CDR residues aredirectly and most substantially involved in influencing antigen binding[see, WO 94/04679 published Mar. 3, 1994].

[0214] Transgenic animals (e.g., mice) that are capable, uponimmunization, of producing a full repertoire of human antibodies in theabsence of endogenous immunoglobulin production can be employed.Transfer of the human germ-line immunoglobulin gene array in suchgerm-line mutant mice will result in the production of human antibodiesupon antigen challenge [see, e.g., Jakobovits et al., Proc. Natl. Acad.Sci. USA, 90:2551-255 (1993); Jakobovits et al., Nature, 362:255-258(1993); Bruggemann et al., Year in Immuno., 7:33 (1993)].

[0215] Human antibodies can also be produced in phage display libraries[Hoogenboom and Winter, J. Mol. Biol., 227:381 (1992); Marks et al., J.Mol. Biol., 222:581 (1991)]. The techniques of Cole et al. and Boerneret al. are also available for the preparation of human monoclonalantibodies (Cole et al., Monoclonal Antibodies and Cancer Therapy, AlanR. Liss, p. 77 (1985) and Boerner et al., J. Immunol., 147(1):86-95(1991)]. Suitable methods for preparing phage libraries have beenreviewed and are described in Winter et al., Annu. Rev. Immunol.,12:433-55 (1994); Soderlind et al., Immunological Reviews, 130:109-123(1992); Hoogenboom, Tibtech February 1997, Vol. 15; Neri et al., CellBiophysics, 27:47-61 (1995). Libraries of single chain antibodies mayalso be prepared by the methods described in WO 92/01047, WO 92/20791,WO 93/06213, WO 93/11236, WO 93/19172, WO 95/01438 and WO 95/15388.Antibody libraries are also commercially available, for example, fromCambridge Antibody Technologies (C.A.T.), Cambridge, UK. Bindingselection against an antigen, in this case Apo-2, can be carried out asdescribed in greater detail in the Examples below.

[0216] As described in the Examples below, anti-Apo-2 single-chain Fv(scFv) antibodies have been identified using a phage display library.Three of these antibodies, referred to herein as 16E2, 24C4 and 20E6,have been sequenced and characterized. The respective DNA and amino acidsequences and complementarity determining regions of these antibodiesare shown in FIGS. 15A-15C and 16. In one embodiment of the invention,scFv Apo-2 antibodies will have the same biological characteristics asthe 16E2, 24C4 or 20E6 antibodies identified herein. The term“biological characteristics” is used to refer to the in vitro and/or invivo activities or properties of the scFv antibody, such as the abilityto specifically bind to Apo-2 or to substantially induce or enhanceApo-2 activation. As disclosed in the present specification, the 16E2,24C4 and 20E6 antibodies are characterized as binding to Apo-2, havingagonistic activity for inducing apoptosis, and having nocross-reactivity to DR4 or several of the other known moleculesrecognized by the Apo-2 ligand. Optionally, the scFv Apo-2 antibody willbind to the same epitope or epitopes recognized by the 16E2, 24C4 or20E6 antibodies disclosed herein. This can be determined by conductingvarious assays, such as described herein and in the Examples. Forinstance, to determine whether a scFv antibody has the same specificityas the 16E2, 24C4 or 20E6 antibodies specifically disclosed, one cancompare activity in apoptosis induction assays, such as those describedin the Examples below.

[0217] Optionally the scFv antibodies to Apo-2 may include antibodieswhich contain a VH and VL chain that include one or more complementaritydetermining region (CDR) amino acid sequences identified in FIG. 16 forthe 16E2, 20E6, or 24C4 antibodies.

[0218] 4. Bispecific Antibodies

[0219] Bispecific antibodies are monoclonal, preferably human orhumanized, antibodies that have binding specificities for at least twodifferent antigens. In the present case, one of the bindingspecificities is for the Apo-2, the other one is for any other antigen,and preferably for a cell-surface protein or receptor or receptorsubunit.

[0220] Methods for making bispecific antibodies are known in the art.Traditionally, the recombinant production of bispecific antibodies isbased on the co-expression of two immunoglobulin heavy-chain/light-chainpairs, where the two heavy chains have different specificities [Milsteinand Cuello, Nature, 305:537-539 (1983)]. Because of the randomassortment of immunoglobulin heavy and light chains, these hybridomas(quadromas) produce a potential mixture of ten different antibodymolecules, of which only one has the correct bispecific structure. Thepurification of the correct molecule is usually accomplished by affinitychromatography steps. Similar procedures are disclosed in WO 93/08829,published May 13, 1993, and in Traunecker et al., EMBO J., 10:3655-3659(1991).

[0221] According to a different and more preferred approach, antibodyvariable domains with the desired binding specificities(antibody-antigen combining sites) are fused to immunoglobulin constantdomain sequences. The fusion preferably is with an immunoglobulinheavy-chain constant domain, comprising at least part of the hinge, CH2,and CH3 regions. It is preferred to have the first heavy-chain constantregion (CH1) containing the site necessary for light-chain bindingpresent in at least one of the fusions. DNAs encoding the immunoglobulinheavy-chain fusions and, if desired, the immunoglobulin light chain, areinserted into separate expression vectors, and are co-transfected into asuitable host organism. This provides for great flexibility in adjustingthe mutual proportions of the three polypeptide fragments in embodimentswhen unequal ratios of the three polypeptide chains used in theconstruction provide the optimum yields. It is, however, possible toinsert the coding sequences for two or all three polypeptide chains inone expression vector when the expression of at least two polypeptidechains in equal ratios results in high yields or when the ratios are ofno particular significance. In a preferred embodiment of this approach,the bispecific antibodies are composed of a hybrid immunoglobulin heavychain with a first binding specificity in one arm, and a hybridimmunoglobulin heavy-chain/light-chain pair (providing a second bindingspecificity) in the other arm. It was found that this asymmetricstructure facilitates the separation of the desired bispecific compoundfrom unwanted immunoglobulin chain combinations, as the presence of animmunoglobulin light chain in only one half of the bispecific moleculeprovides for a facile way of separation. This approach is disclosed inWO 94/04690 published Mar. 3, 1994. For further details of generatingbispecific antibodies see, for example, Suresh et al., Methods inEnzymology, 121:210 (1986).

[0222] 5. Heteroconjugate Antibodies

[0223] Heteroconjugate antibodies are also within the scope of thepresent invention. Heteroconjugate antibodies are composed of twocovalently joined antibodies. Such antibodies have, for example, beenproposed to target immune system cells to unwanted cells [U.S. Pat. No.4,676,980], and for treatment of HIV infection [WO 91/00360; WO92/200373; EP 03089]. It is contemplated that the antibodies may beprepared in vitro using known methods in synthetic protein chemistry,including those involving crosslinking agents. For example, immunotoxinsmay be constructed using a disulfide exchange reaction or by forming athioether bond. Examples of suitable reagents for this purpose includeiminothiolate and methyl-4-mercaptobutyrimidate and those disclosed, forexample, in U.S. Pat. No. 4,676,980.

[0224] 6. Triabodies

[0225] Triabodies are also within the scope of the invention. Suchantibodies are described for instance in Iliades et al., FEBS Letters,409:437-441 (1997) and Korrt et al., Protein Engineering, 10:423-433(1997).

[0226] 7. Other Modifications

[0227] Other modifications of the Apo-2 antibodies are contemplated. Forexample, it may be desirable to modify the antibodies of the inventionwith respect to effector function, so as to enhance the therapeuticeffectiveness of the antibodies. For instance, cysteine residue(s) maybe introduced into the Fc region, thereby allowing interchain disulfidebond formation in this region. The homodimeric antibody thus generatedmay have improved internalization capability and/or increasedcomplement-mediated cell killing [see, e.g., Caron et al., J. Exp. Med.,176:1191-1195 (1992); Shopes, J. Immunol., 148:2918-2922 (1992).Homodimeric antibodies may also be prepared using heterobifunctionalcross-linkers as described in Wolff et al., Cancer Research,53:2560-2565 (1993). Ghetie et al., Proc. Natl. Acad. Sci., 94:7509-7514(1997), further describe preparation of IgG-IgG homodimers and disclosethat such homodimers can enhance apoptotic activity as compared to themonomers. Alternatively, the antibodies can be engineered to have dualFc regions [see, Stevenson et al., Anti-Cancer Drug Design, 3:219-230(1989)].

[0228] It may be desirable to modify the amino acid sequences of theantibodies disclosed herein. Sequences within the scFv complementarydetermining or linker regions (as shown in FIG. 16) may be modified forinstance to modulate the biological activities of these antibodies.Variations in the full-length scFv sequence or in various domains of thescFv molecules described herein, can be made, for example, using any ofthe techniques and guidelines for conservative and non-conservativemutations set forth, for instance, in U.S. Pat. No. 5,364,934.Variations may be a substitution, deletion or insertion of one or morecodons encoding a scFv that results in a change in the amino acidsequence of the scFv as compared with the native sequence scFv.Optionally, the variation is by substitution of at least one amino acidwith any other amino acid in one or more of the domains of the scFvmolecule. The variations can be made using methods known in the art suchas oligonucleotide-mediated (site-directed) mutagenesis, alaninescanning, and PCR mutagenesis. Site-directed mutagenesis [Carter et al.,Nucl. Acids Res., 13:4331 (1986); Zoller et al., Nucl. Acids Res.,10:6487 (1987)], cassette mutagenesis [Wells et al., Gene, 34:315(1985)], restriction selection mutagenesis [Wells et al., Philos. Trans.R. Soc. London SerA, 317:415 (1986)] or other known techniques can beperformed on the cloned DNA to produce the scFv variant DNA.

[0229] The antibodies may optionally be covalently attached orconjugated to one or more chemical groups. A polyol, for example, can beconjugated to an antibody molecule at one or more amino acid residues,including lysine residues as disclosed in WO 93/00109. Optionally, thepolyol is a poly(alkelene glycol), such as poly(ethylene glycol) (PEG),however, those skilled in the art recognize that other polyols, such as,for example, poly(propylene glycol) and polyethylene-polypropyleneglycol copolymers, can be employed using techniques for conjugating PEGto polypeptides. A variety of methods for pegylating polypeptides havebeen described. See, e.g. U.S. Pat. No. 4,179,337 which discloses theconjugation of a number of hormones and enzymes to PEG and polypropyleneglycol to produce physiologically active compositions having reducedimmunogenicities.

[0230] The antibodies may also be fused or linked to anotherheterologous polypeptide or amino acid sequence such as an epitope tag.Epitope tag polypeptides and methods of their use are described above inSection A, paragraph 8. Any of the tags described herein may be linkedto the antibodies. The Examples below, for instance, describe His-taggedand gD-tagged single-chain antibodies.

[0231] D. Therapeutic Uses for Apo-2 Antibodies

[0232] The Apo-2 antibodies of the invention have therapeutic utility.Agonistic Apo-2 antibodies, for instance, may be employed to activate orstimulate apoptosis in cancer cells. Accordingly, the invention providesmethods for treating cancer using such Apo-2 antibodies. It is of coursecontemplated that the methods of the invention can be employed incombination with still other therapeutic techniques such as surgery.

[0233] The agonist is preferably administered to the mammal in acarrier. Suitable carriers and their formulations are described inRemington's Pharmaceutical Sciences, 16th ed., 1980, Mack PublishingCo., edited by Oslo et al. Typically, an appropriate amount of apharmaceutically-acceptable salt is used in the formulation to renderthe formulation isotonic. Examples of a pharmaceutically-acceptablecarrier include saline, Ringer's solution and dextrose solution. The pHof the solution is preferably from about 5 to about 8, and morepreferably from about 7 to about 7.5. Further carriers include sustainedrelease preparations such as semipermeable matrices of solid hydrophobicpolymers containing the agonist, which matrices are in the form ofshaped articles, e.g., films, liposomes or microparticles. It will beapparent to those persons skilled in the art that certain carriers maybe more preferable depending upon, for instance, the route ofadministration and concentration of agonist being administered.

[0234] The agonist antibody can be administered to the mammal byinjection (e.g., intravenous, intraperitoneal, subcutaneous,intramuscular), or by other methods such as infusion that ensure itsdelivery to the bloodstream in an effective form. The agonist may alsobe administered by intratumoral, peritumoral, intralesional, orperilesional routes, to exert local as well as systemic therapeuticeffects. Local or intravenous injection is preferred.

[0235] Effective dosages and schedules for administering the agonistantibody may be determined empirically, and making such determinationsis within the skill in the art. Those skilled in the art will understandthat the dosage of agonist that must be administered will vary dependingon, for example, the mammal which will receive the agonist, the route ofadministration, the particular type of agonist used and other drugsbeing administered to the mammal. Guidance in selecting appropriatedoses for antibody agonists is found in the literature on therapeuticuses of antibodies, e.g., Handbook of Monoclonal Antibodies, Ferrone etal., eds., Noges Publications, Park Ridge, N.J., (1985) ch. 22 and pp.303-357; Smith et al., Antibodies in Human Diagnosis and Therapy, Haberet al., eds., Raven Press, New York (1977) pp. 365-389. A typical dailydosage of the agonist used alone might range from about 1 μg/kg to up to100 mg/kg of body weight or more per day, depending on the factorsmentioned above.

[0236] The agonist antibody may also be administered to the mammal incombination with effective amounts of one or more other therapeuticagents or in conjunction with radiation treatment. Therapeutic agentscontemplated include chemotherapeutics as well as immunoadjuvants andcytokines. Chemotherapies contemplated by the invention include chemicalsubstances or drugs which are known in the art and are commerciallyavailable, such as Doxorubicin, 5-Fluorouracil, Cytosine arabinoside(“Ara-C”), Cyclophosphamide, Thiotepa, Busulfan, Cytoxin, Taxol,Methotrexate, Cisplatin, Melphalan, Vinblastine and Carboplatin. Theagonist may be administered sequentially or concurrently with the one ormore other therapeutic agents. The amounts of agonist and therapeuticagent depend, for example, on what type of drugs are used, the cancerbeing treated, and the scheduling and routes of administration but wouldgenerally be less than if each were used individually.

[0237] Following administration of agonist to the mammal, the mammal'scancer and physiological condition can be monitored in various ways wellknown to the skilled practitioner. For instance, tumor mass may beobserved physically or by standard x-ray imaging techniques.

[0238] The Apo-2 antibodies of the invention may also be useful inenhancing immune-mediated cell death in cells expressing Apo-2, forinstance, through complement fixation or ADCC. Alternatively,antagonistic antibodies may be used to block excessive apoptosis (forinstance in neurodegenerative disease) or to block potentialautoimmune/inflammatory effects of Apo-2 resulting from NF-κBactivation. Such antagonistic antibodies can be utilized according tothe therapeutic methods and techiques described above.

[0239] E. Non-therapeutic Uses for Apo-2 Antibodies

[0240] Apo-2 antibodies may further be used in diagnostic assays forApo-2, e.g., detecting its expression in specific cells, tissues, orserum. Various diagnostic assay techniques known in the art may be used,such as competitive binding assays, direct or indirect sandwich assaysand immunoprecipitation assays conducted in either heterogeneous orhomogeneous phases [Zola, Monoclonal Antibodies: A Manual of Techniques,CRC Press, Inc. (1987) pp. 147-158]. The antibodies used in thediagnostic assays can be labeled with a detectable moiety. Thedetectable moiety should be capable of producing, either directly orindirectly, a detectable signal. For example, the detectable moiety maybe a radioisotope, such as ³H, ¹⁴C, ³²P, ³⁵S, or ¹²⁵I, a fluorescent orchemiluminescent compound, such as fluorescein isothiocyanate,rhodamine, or luciferin, or an enzyme, such as alkaline phosphatase,beta-galactosidase or horseradish peroxidase. Any method known in theart for conjugating the antibody to the detectable moiety may beemployed, including those methods described by Hunter et al., Nature,144:945 (1962); David et al., Biochemistry, 13:1014 (1974); Pain et al.,J. Immunol. Meth., 40:219 (1981); and Nygren, J. Histochem. andCytochem., 30:407 (1982).

[0241] Apo-2 antibodies also are useful for the affinity purification ofApo-2 from recombinant cell culture or natural sources. In this process,the antibodies against Apo-2 are immobilized on a suitable support, suchas Sephadex resin or filter paper, using methods well known in the art.The immobilized antibody then is contacted with a sample containing theApo-2 to be purified, and thereafter the support is washed with asuitable solvent that will remove substantially all the material in thesample except the Apo-2, which is bound to the immobilized antibody.Finally, the support is washed with another suitable solvent that willrelease the Apo-2 from the antibody.

[0242] F. Kits Containing Apo-2 or Apo-2 Antibodies

[0243] In a further embodiment of the invention, there are providedarticles of manufacture and kits containing Apo-2 or Apo-2 antibodieswhich can be used, for instance, for the therapeutic or non-therapeuticapplications described above. The article of manufacture comprises acontainer with a label. Suitable containers include, for example,bottles, vials, and test tubes. The containers may be formed from avariety of materials such as glass or plastic. The container holds acomposition which includes an active agent that is effective fortherapeutic or non-therapeutic applications, such as described above.The active agent in the composition is Apo-2 or an Apo-2 antibody. Thelabel on the container indicates that the composition is used for aspecific therapy or non-therapeutic application, and may also indicatedirections for either in vivo or in vitro use, such as those describedabove.

[0244] The kit of the invention will typically comprise the containerdescribed above and one or more other containers comprising materialsdesirable from a commercial and user standpoint, including buffers,diluents, filters, needles, syringes, and package inserts withinstructions for use.

[0245] The following examples are offered for illustrative purposesonly, and are not intended to limit the scope of the present inventionin any way.

[0246] All patent and literature references cited in the presentspecification are hereby incorporated by reference in their entirety.

EXAMPLES

[0247] All restriction enzymes referred to in the examples werepurchased from New England Biolabs and used according to manufacturer'sinstructions. All other commercially available reagents referred to inthe examples were used according to manufacturer's instructions unlessotherwise indicated. The source of those cells identified in thefollowing examples, and throughout the specification, by ATCC accessionnumbers is the American Type Culture Collection, Manassas, Va.

Example 1

[0248] Isolation of cDNA clones Encoding Human Apo-2

[0249] Expressed sequence tag (EST) DNA databases (LIFESEQ™, IncytePharmaceuticals, Palo Alto, Calif.) were searched and an EST wasidentified which showed homology to the death domain of the Apo-3receptor [Marsters et al., Curr. Biol., 6:750 (1996)]. Human pancreasand kidney lgt10 bacteriophage cDNA libraries (both purchased fromClontech) were ligated into pRK5 vectors as follows. Reagents were addedtogether and incubated at 16° C. for 16 hours: 5×T4 ligase buffer (3ml); pRK5, XhoI, NotI digested vector, 0.5 mg, 1 ml); cDNA (5 ml) anddistilled water (6 ml). Subsequently, additional distilled water (70 ml)and 10 mg/ml tRNA (0.1 ml) were added and the entire reaction wasextracted through phenol:chloroform:isoamyl alcohol (25:24:1). Theaqueous phase was removed, collected and diluted into 5M NaCl (10 ml)and absolute ethanol (−20° C., 250 ml). This was then centrifuged for 20minutes at 14,000×g, decanted, and the pellet resuspended into 70%ethanol (0.5 ml) and centrifuged again for 2 minutes at 14,000×g. TheDNA pellet was then dried in a speedvac and eluted into distilled water(3 ml) for use in the subsequent procedure.

[0250] The ligated cDNA/pRK5 vector DNA prepared previously was chilledon ice to which was added electrocompetent DH10B bacteria (Life Tech.,20 ml). The bacteria vector mixture was then electroporated as per themanufacturers recommendation. Subsequently SOC media (1 ml) was addedand the mixture was incubated at 37° C. for 30 minutes. Thetransformants were then plated onto 20 standard 150 mm LB platescontaining ampicillin and incubated for 16 hours (37° C.) to allow thecolonies to grow. Positive colonies were then scraped off and the DNAisolated from the bacterial pellet using standard CsCl-gradientprotocols.

[0251] An enriched 5′-cDNA library was then constructed to obtain a biasof cDNA fragments which preferentially represents the 5′ ends of cDNA'scontained within the library. 10 mg of the pooled isolated full-lengthlibrary plasmid DNA (41 ml) was combined with Not 1 restriction buffer(New England Biolabs, 5 ml) and Not 1 (New England Biolabs, 4 ml) andincubated at 37° C. for one hour. The reaction was extracted throughphenol:chloroform:isoamyl alcohol (25:24:1, 50 ml), the aqueous phaseremoved, collected and resuspended into 5M NaCl (5 ml) and absoluteethanol (−20° C., 150 ml). This was then centrifuged for 20 minutes at14,000×g, decanted, resuspended into 70% ethanol (0.5 ml) andcentrifuged again for 2 minutes at 14,000×g. The supernatant was thenremoved, the pellet dried in a speedvac and resuspended in distilledwater (10 ml).

[0252] The following reagents were brought together and incubated at 37°C. for 2 hours: distilled water (3 ml); linearized DNA library (1 mg, 1ml); Ribonucleotide mix (Invitrogen, 10 ml); transcription buffer(Invitrogen, 2 ml) and Sp6 enzyme mix. The reaction was then extractedthrough phenol:chloroform:isoamyl alcohol (25:24:1, 50 ml) and theaqueous phase was removed, collected and resuspended into 5M NaCl (5 ml)and absolute ethanol (−20° C., 150 ml) and centrifuged for 20 minutes at14,000×g. The pellet was then decanted and resuspended in 70% ethanol(0.5 ml), centrifuged again for 2 minutes at 14,000×g, decanted, driedin a speedvac and resuspended into distilled water (10 ml).

[0253] The following reagents were added together and incubated at 16°C. for 16 hours: 5×T4 ligase buffer (Life Tech., 3 ml); pRK5 Cla-Saldigested vector, 0.5 mg, 1 ml); cDNA (5 ml); distilled water (6 ml).Subsequently, additional distilled water (70 ml) and 10 mg/ml tRNA (0.1ml) was added and the entire reaction was extracted throughphenol:chloroform:isoamyl alcohol (25:24:1, 100 ml). The aqueous phasewas removed, collected and diluted by 5M NaCl (10 ml) and absoluteethanol (−20° C., 250 ml) and centrifuged for 20 minutes at 14,000×g.The DNA pellet was decanted, resuspended into 70% ethanol (0.5 ml) andcentrifuged again for 2 minutes at 14,000×g. The supernatant was removedand the residue pellet was dried in a speedvac and resuspended indistilled water (3 ml). The ligated cDNA/pSST-amy.1 vector DNA waschilled on ice to which was added electrocompetent DH10B bacteria (LifeTech., 20 ml). The bacteria vector mixture was then electroporated asrecommended by the manufacturer. Subsequently, SOC media (Life Tech., 1ml) was added and the mixture was incubated at 37° C. for 30 minutes.The transformants were then plated onto 20 standard 150 mm LB platescontaining ampicillin and incubated for 16 hours (37° C.). Positivecolonies were scraped off the plates and the DNA was isolated from thebacterial pellet using standard protocols, e.g. CsCl-gradient.

[0254] The cDNA libraries were screened by hybridization with asynthetic oligonucleotide probe:

[0255]GGGAGCCGCTCATGAGGAAGTTGGGCCTCATGGACAATGAGATAAAGGTGGCTAAAGCTGAGGCAGC GGG(SEQ ID NO:3) based on the EST.

[0256] Three cDNA clones were sequenced in entirety. The overlappingcoding regions of the cDNAs were identical except for codon 410 (usingthe numbering system for FIG. 1); this position encoded a leucineresidue (TTG) in both pancreatic cDNAs, and a methionine residue (ATG)in the kidney cDNA, possibly due to polymorphism.

[0257] The entire nucleotide sequence of Apo-2 is shown in FIG. 1 (SEQID NO:2). Clone 27868 (also referred to as pRK5-Apo-2 deposited as ATCC209021, as indicated below) contains a single open reading frame with anapparent translational initiation site at nucleotide positions 140-142[Kozak et al., supra] and ending at the stop codon found at nucleotidepositions 1373-1375 (FIG. 1; SEQ ID NO:2). The predicted polypeptideprecursor is 411 amino acids long, a type I transmembrane protein, andhas a calculated molecular weight of approximately 45 kDa. Hydropathyanalysis (not shown) suggested the presence of a signal sequence(residues 1-53), followed by an extracellular domain (residues 54-182),a transmembrane domain (residues 183-208), and an intracellular domain(residues 209-411) (FIG. 2A; SEQ ID NO:1). N-terminal amino acidsequence analysis of Apo-2-IgG expressed in 293 cells showed that themature polypeptide starts at amino acid residue 54, indicating that theactual signal sequence comprises residues 1-53. Apo-2 polypeptide isobtained or obtainable by expressing the molecule encoded by the cDNAinsert of the deposited ATCC 209021 vector.

[0258] TNF receptor family proteins are typically characterized by thepresence of multiple (usually four) cysteine-rich domains in theirextracellular regions—each cysteine-rich domain being approximately 45amino acids long and containing approximately 6, regularly spaced,cysteine residues. Based on the crystal structure of the type 1 TNFreceptor, the cysteines in each domain typically form three disulfidebonds in which usually cysteines 1 and 2, 3 and 5, and 4 and 6 arepaired together. Like DR4, Apo-2 contains two extracellularcysteine-rich pseudorepeats (FIG. 2A), whereas other identifiedmammalian TNFR family members contain three or more such domains [Smithet al., Cell, 76:959 (1994)].

[0259] The cytoplasmic region of Apo-2 contains a death domain (aminoacid residues 324-391 shown in FIG. 1; see also FIG. 2A) which showssignificantly more amino acid sequence identity to the death domain ofDR4 (64%) than to the death domain of TNFR1 (30%); CD95 (19%); orApo-3/DR3 (29%) (FIG. 2B). Four out of six death domain amino acids thatare required for signaling by TNFR1 [Tartaglia et al., supra] areconserved in Apo-2 while the other two residues are semi-conserved (seeFIG. 2B).

[0260] Based on an alignment analysis (using the ALIGN™ computerprogram) of the full-length sequence, Apo-2 shows more sequence identityto DR4 (55%) than to other apoptosis-linked receptors, such as TNFR1(19%); CD95 (17%); or Apo-3 (also referred to as DR3, WSL-1 or TRAMP)(29%).

Example 2

[0261] A. Expression of Apo-2 ECD

[0262] A soluble extracellular domain (ECD) fusion construct wasprepared. An Apo-2 ECD (amino acid residues 1-184 shown in FIG. 1) wasobtained by PCR and fused to a C-terminal Flag epitope tag (Sigma). (TheApo-2 ECD construct included residues 183 and 184 shown in FIG. 1 toprovide flexibility at the junction, even though residues 183 and 184are predicted to be in the transmembrane region). The Flagepitope-tagged molecule was then inserted into pRK5, and expressed bytransient transfection into human 293 cells (ATCC CRL 1573).

[0263] After a 48 hour incubation, the cell supernatants were collectedand either used directly for co-precipitation studies (see Example 3) orsubjected to purification of the Apo-2 ECD-Flag by affinitychromatography on anti-Flag agarose beads, according to manufacturer'sinstructions (Sigma).

[0264] B. Expression of Apo-2 ECD as an Immunoadhesin

[0265] A soluble Apo-2 ECD immunoadhesin construct was prepared. TheApo-2 ECD (amino acids 1-184 shown in FIG. 1) was fused to the hinge andFc region of human immunoglobulin G₁ heavy chain in pRK5 as describedpreviously [Ashkenazi et al., Proc. Natl. Acad. Sci., 88:10535-10539(1991)]. The immunoadhesin was expressed by transient transfection intohuman 293 cells and purified from cell supernatants by protein Aaffinity chromatography, as described by Ashkenazi et al., supra.

Example 3

[0266] Immunoprecipitation Assay Showing Binding Interaction BetweenApo-2 and Apo-2 Ligand

[0267] To determine whether Apo-2 and Apo-2L interact or associate witheach other, supernatants from mock-transfected 293 cells or from 293cells transfected with Apo-2 ECD-Flag (described in Example 2 above) (5ml) were incubated with 5 μg poly-histidine-tagged soluble Apo-2L [Pittiet al., supra] for 30 minutes at room temperature and then analyzed forcomplex formation by a co-precipitation assay.

[0268] The samples were subjected to immunoprecipitation using 25 μlanti-Flag conjugated agarose beads (Sigma) or Nickel-conjugated agarosebeads (Qiagen). After a 1.5 hour incubation at 4° C., the beads werespun down and washed four times in phosphate buffered saline (PBS). Byusing anti-Flag agarose, the Apo-2L was precipitated through theFlag-tagged Apo-2 ECD; by using Nickel-agarose, the Apo-2 ECD wasprecipitated through the His-tagged Apo-2L. The precipitated proteinswere released by boiling the beads for 5 minutes in SDS-PAGE buffer,resolved by electrophoresis on 12% polyacrylamide gels, and thendetected by immunoblot with anti-Apo-2L or anti-Flag antibody (2 μg/ml)as described in Marsters et al., J. Biol. Chem., (1997).

[0269] The results, shown in FIG. 3, indicate that the Apo-2 ECD andApo-2L can associate with each other.

[0270] The binding interaction was further analyzed by purifying Apo-2ECD from the transfected 293 cell supernatants with anti-Flag beads (seeExample 2) and then analyzing the samples on a BIACORE™ instrument. TheBIACORE™ analysis indicated a dissociation constant (K_(d)) of about 1nM. BIACORE™ analysis also showed that the Apo-2 ECD is not capable ofbinding other apoptosis-inducing TNF family members, namely, TNF-alpha(Genentech, Inc., Pennica et al., Nature, 312:712 (1984),lymphotoxin-alpha (Genentech, Inc.), or Fas/Apo-1 ligand (AlexisBiochemicals). The data thus shows that Apo-2 is a specific receptor forApo-2L.

Example 4

[0271] Induction of Apoptosis by Apo-2

[0272] Because death domains can function as oligomerization interfaces,over-expression of receptors that contain death domains may lead toactivation of signaling in the absence of ligand [Frazer et al., supra,Nagata et al., supra]. To determine whether Apo-2 was capable ofinducing cell death, human 293 cells or HeLa cells (ATCC CCL 2.2) weretransiently transfected by calcium phosphate precipitation (293 cells)or electroporation (HeLa cells) with a pRK5 vector or pRK5-basedplasmids encoding Apo-2 and/or CrmA. When applicable, the total amountof plasmid DNA was adjusted by adding vector DNA. Apoptosis was assessed24 hours after transfection by morphology (FIG. 4A); DNA fragmentation(FIG. 4B); or by FACS analysis of phosphatydilserine exposure (FIG. 4C)as described in Marsters et al., Curr. Biol., 6: 1669 (1996). As shownin FIGS. 4A and 4B, the Apo-2 transfected 293 cells underwent markedapoptosis.

[0273] For samples assayed by FACS, the HeLa cells were co-transfectedwith pRK5-CD4 as a marker for transfection and apoptosis was determinedin CD4-expressing cells; FADD was co-transfected with the Apo-2 plasmid;the data are means±SEM of at least three experiments, as described inMarsters et al., Curr. Biol., 6:1669 (1996). The caspase inhibitors,DEVD-fmk (Enzyme Systems) or z-VAD-fmk (Research Biochemicals Intl.)were added at 200 μM at the time of transfection. As shown in FIG. 4C,the caspase inhibitors CrmA, DEVD-fmk, and z-VAD-fmk blocked apoptosisinduction by Apo-2, indicating the involvement of Ced-3-like proteasesin this response.

[0274] FADD is an adaptor protein that mediates apoptosis activation byCD95, TNFR1, and Apo-3/DR3 [Nagata et al., supra], but does not appearnecessary for apoptosis induction by Apo-2L [Marsters et al., supra] orby DR4 [Pan et al., supra]. A dominant-negative mutant form of FADD,which blocks apoptosis induction by CD95, TNFR1, or Apo-3/DR3 [Frazer etal., supra; Nagata et al., supra; Chinnayian et al., supra] did notinhibit apoptosis induction by Apo-2 when co-transfected into HeLa cellswith Apo-2 (FIG. 4C). These results suggest that Apo-2 signals apoptosisindependently of FADD. Consistent with this conclusion, aglutathione-S-transferase fusion protein containing the Apo-2cytoplasmic region did not bind to in vitro transcribed and translatedFADD (data not shown).

Example 5

[0275] Inhibition of Apo-2L Activity by Soluble Apo-2 ECD

[0276] Soluble Apo-2L (0.5 μg/ml, prepared as described in Pitti et al.,supra) was pre-incubated for 1 hour at room temperature with PBS bufferor affinity-purified Apo-2 ECD (5 μg/ml) together with anti-Flagantibody (Sigma) (1 μg/ml) and added to HeLa cells. After a 5 hourincubation, the cells were analyzed for apoptosis by FACS (as above)(FIG. 4D).

[0277] Apo-2L induced marked apoptosis in HeLa cells, and the solubleApo-2 ECD was capable of blocking Apo-2L action (FIG. 4D), confirming aspecific interaction between Apo-2L and Apo-2. Similar results wereobtained with the Apo-2 ECD immunoadhesin (FIG. 4D). Dose-responseanalysis showed half-maximal inhibition at approximately 0.3 nM Apo-2immunoadhesin (FIG. 4E).

Example 6

[0278] Activation of NF-κB by Apo-2

[0279] An assay was conducted to determine whether Apo-2 activatesNF-κB.

[0280] HeLa cells were transfected with pRK5 expression plasmidsencoding full-length native sequence Apo-2, DR4 or Apo-3 and harvested24 hours after transfection. Nuclear extracts were prepared and 1 μg ofnuclear protein was reacted with a ³²P-labelled NF-κB-specific syntheticoligonucleotide probe ATCAGGGACTTTCCGCTGGGGACTTTCCG (SEQ ID NO:4) [see,also, MacKay et al., J. Immunol., 153:5274-5284 (1994)], alone ortogether with a 50-fold excess of unlabelled probe, or with anirrelevant ³²P-labelled synthetic oligonucleotideAGGATGGGAAGTGTGTGATATATCCTTGAT (SEQ ID NO:5). In some samples, antibodyto p65/RelA subunits of NF-κB (1 μg/ml; Santa Cruz Biotechnology) wasadded. DNA binding was analyzed by an electrophoretic mobility shiftassay as described by Hsu et al., supra; Marsters et al., supra, andMacKay et al., supra.

[0281] The results are shown in FIG. 5. As shown in FIG. 5A, upontransfection into HeLa cells, both Apo-2 and DR4 induced significantNF-κB activation as measured by the electrophoretic mobility shiftassay; the level of activation was comparable to activation observed forApo-3/DR3. Antibody to the p65/RelA subunit of NF-κB inhibited themobility of the NF-κB probe, implicating p65 in the response to all 3receptors.

[0282] An assay was also conducted to determine if Apo-2L itself canregulate NF-κB activity. HeLa cells or MCF7 cells (human breastadenocarcinoma cell line, ATCC HTB 22) were treated with PBS buffer,soluble Apo-2L (Pitti et al., supra) or TNF-alpha (Genentech, Inc., seePennica et al., Nature, 312:721 (1984)) (1 μg/ml) and assayed for NF-κBactivity as above. The results are shown in FIG. 5B. The Apo-2L induceda significant NF-KB activation in the treated HeLa cells but not in thetreated MCF7 cells; the TNF-alpha induced a more pronounced activationin both cell lines. Several studies have disclosed that NF-κB activationby TNF can protect cells against TNF-induced apoptosis [Nagata, supra].

[0283] The effects of a NF-KB inhibitor, ALLN(N-acetyl-Leu-Leu-norleucinal) and a transcription inhibitor,cyclohexamide, were also tested. The HeLa cells (plated in 6-welldishes) were preincubated with PBS buffer, ALLN (Calbiochem) (40 μg/ml)or cyclohexamide (Sigma) (50 μg/ml) for 1 hour before addition of Apo-2L(1 μg/ml). After a 5 hour incubation, apoptosis was analyzed by FACS(see FIG. 5C).

[0284] The results are shown in FIG. 5C. Both ALLN and cyclohexamideincreased the level of Apo-2L-induced apoptosis in the HeLa cells. Thedata indicates that Apo-2L can induce protective NF-κB-dependent genes.The data also indicates that Apo-2L is capable of activating NF-κB incertain cell lines and that both Apo-2 and DR4 may mediate thatfunction.

Example 7

[0285] Expression of Apo-2 in Mammalian Tissues

[0286] A. Northern Blot Analysis

[0287] Expression of Apo-2 mRNA in human tissues was examined byNorthern blot analysis. Human RNA blots were hybridized to a 4.6kilobase ³²P-labelled DNA probe based on the full length Apo-2 cDNA; theprobe was generated by digesting the pRK5-Apo-2 plasmid with EcoRI.Human fetal RNA blot MTN (Clontech), human adult RNA blot MTN-II(Clontech), and human cancer cell line RNA blot (Clontech) wereincubated with the DNA probes. Blots were incubated with the probes inhybridization buffer (5×SSPE; 2× Denhardt's solution; 100 mg/mLdenatured sheared salmon sperm DNA; 50% formamide; 2% SDS) for 60 hoursat 42° C. The blots were washed several times in 2×SSC; 0.05% SDS for 1hour at room temperature, followed by a 30 minute wash in 0.1×SSC; 0.1%SDS at 50° C. The blots were developed after overnight exposure.

[0288] As shown in FIG. 6A, a predominant mRNA transcript ofapproximately 4.6 kb was detected in multiple tissues. Expression wasrelatively high in fetal and adult liver and lung, and in adult ovaryand peripheral blood leukocytes (PBL), while no mRNA expression wasdetected in fetal and adult brain. Intermediate levels of expressionwere seen in adult colon, small intestine, testis, prostate, thymus,pancreas, kidney, skeletal muscle, placenta, and heart. Several adulttissues that express Apo-2, e.g., PBL, ovary, and spleen, have beenshown previously to express DR4 [Pan et al., supra], however, therelative levels of expression of each receptor mRNA appear to bedifferent.

[0289] As shown in FIG. 6B, Apo-2 mRNA was expressed relatively high in6 of 8 human cancer cell lines examined, namely, HL60 promyelocyticleukemia, HeLa S3 cervical carcinoma, K562 chronic myelogenous leukemia,SW 480 colorectal adenocarcinoma, A549 lung carcinoma, and G361melanoma. There was also detectable expression in Burkitt's lymphoma(Raji) cells. Thus, Apo-2 may be useful as a target for inducingapoptosis in cancer cells from lymphoid as well as non-lymphoid tumors.

[0290] B. In Situ Hybridization

[0291] Expression of Apo-2 in normal and in cancerous human tissues wasexamined by in situ hybridization. In addition, several different chimpand rhesus monkey tissues were examined for Apo-2 expression. Thesetissues included: human fetal tissues (E12-E16 weeks)—placenta,umbilical cord, liver, kidney, adrenal gland, thyroid, lung, heart,great vessels, esophagus, stomach, small intestine, spleen, thymus,pancreas, brain, eye, spinal cord, body wall, pelvis and lower limb;adult human tissues—kidney, bladder, adrenal gland, spleen, lymph node,pancreas, lung, skin, retina, liver; chimp tissues—salivary gland,stomach, thyroid, parathyroid, tongue, thymus, ovary, lymph node, andperipheral nerve; rhesus monkey tissues—cerebral cortex, hippocampus,cerebellum and penis; human tumor tissue—lung adenocarcinoma, testis,lung carcinoma, breast carcinoma, fibroadenoma, soft tissue sarcoma.

[0292] Tissue samples were paraffin-embedded and sectioned. Later, thesectioned tissues were deparaffinized and the slides placed in water.The slides were rinsed twice for five minutes at room temperature in2×SSC. After rinsing, the slides were placed in 20 μg/ml proteinase K(in Rnase-free buffer) for 15 minutes at 37° C. (for fetal tissues) or8× proteinase K for 30 minutes at 37° C. (for formalin tissues). Theslides were then rinsed again in 0.5× SSC and dehydrated. Prior tohybridization, the slides were placed in a plastic box lined with buffer(4×SSC, 50% formamide)—saturated filter paper. The tissues were coveredwith 50 μl hybridization buffer (3.75 g Dextran sulfate plus 6 ml water;vortexed and heated for 2 minutes; cooled on ice and 18.75 ml formamide,3.75 ml 20×SSC and 9 ml water added) and incubated at 42° C. for 1 to 4hours.

[0293] Hybridization was conducted using a ³³P labelled probe consistingof nucleotides 706-1259 of SEQ ID NO:2. The probe was added to theslides in hybridization buffer and incubated overnight at 55° C.Multiple washing steps were then performed sequentially as follows:twice for 10 minutes at room temperature in 2×SSC, EDTA buffer (400 ml20×SSC, 16 ml 0.25M EDTA); once for 30 minutes at 37° C. in 20 μg/mlRNase A; twice for 10 minutes at room temperature in 2×SSC, EDTA buffer;once for 2 hours at 55° C. in 0.1×SSC, EDTA buffer; twice for 10 minutesat room temperature in 0.5×SSC. Dehydration was performed for 2 minuteseach in 50%, 70%, 90% EtOH containing 0.3 M NH₄AC. Finally, the slideswere air-dried for 2 hours and exposed to film.

[0294] Expression of Apo-2 in the fetal tissues appeared strongest overhepatocytes in liver, developing glomeruli in kidney, adrenal cortex,and epithelium of gastrointestinal tract. Moderate expression wasobserved over epithelial cells in lung and at sites of vascularizationof a bone growth plate. A relatively low level expression was observedover thyroid epithelial cells and cells in cardiac ventricles.Expression was observed over lymphoid cells in the thymic medulla,developing lymph glands and placenta cytotrophoblast cells.

[0295] Expression of Apo-2 in adult tissues was observed over restingoocytes in primordial follicles and low levels over granulosa cells ofdeveloping follicles in chimp ovary. Expression was observed incirrhotic livers over hepatocytes at the edge of nodules (i.e., area ofdamage, normal adult liver was negative). Other tissues were negativefor expression.

[0296] In the cancer tissues examined, Apo-2 expression was found in twolung adenocarcinomas and two germ cell tumors of the testis. Twoadditional lung carcinomas (one squamous) were negative. One of fivebreast carcinomas was positive (there was expression in normal breasttissue). In a fibroadenoma, there appeared to be expression over bothepithelial and stromal elements. A soft tissue sarcoma was alsopositive. Other tissues examined were negative.

Example 8

[0297] Chromosomal Localization of the Apo-2 Gene

[0298] Chromosomal localization of the human Apo-2 gene was examined byradiation hybrid (RH) panel analysis. RH mapping was performed by PCRusing a human-mouse cell radiation hybrid panel (Research Genetics) andprimers based on the coding region of the Apo-2 cDNA [Gelb et al., Hum.Genet., 98:141 (1996)). Analysis of the PCR data using the StanfordHuman Genome Center Database indicates that Apo-2 is linked to themarker D8S481, with an LOD of 11.05; D8S481 is linked in turn toD8S2055, which maps to human chromosome 8p21. A similar analysis of DR4showed that DR4 is linked to the marker D8S2127 (with an LOD of 13.00),which maps also to human chromosome 8p21.

[0299] To Applicants' present knowledge, to date, no other member of theTNFR gene family has been located to chromosome 8.

Example 9

[0300] Preparation of Monoclonal Antibodies Specific for Apo-2

[0301] Balb/c mice (obtained from Charles River Laboratories) wereimmunized by injecting 0.5 μg/50 μl of an Apo-2 ECD immunoadhesinprotein (diluted in MPL-TDM adjuvant purchased from Ribi ImmunochemicalResearch Inc., Hamilton, Mont.) 11 times into each hind foot pad at 3-4day intervals. The Apo-2 ECD immunoadhesin protein was generated byfusing an extracellular domain sequence of Apo-2 (amino acids 1-184shown in FIG. 1) to the hinge and Fc region of human immunoglobulin G₁heavy chain in pRK5 as described previously [Ashkenazi et al., Proc.Natl. Acad. Sci., 88:10535-10539 (1991)]. The immunoadhesin protein wasexpressed by transient transfection into human 293 cells and purifiedfrom cell supernatants by protein A affinity chromatography, asdescribed by Ashkenazi et al., supra (See also Example 2B above).

[0302] Three days after the final boost, popliteal lymph nodes wereremoved from the mice and a single cell suspension was prepared in DMEMmedia (obtained from Biowhitakker Corp.) supplemented with 1%penicillin-streptomycin. The lymph node cells were then fused withmurine myeloma cells P3X63AgU.1 (ATCC CRL 1597) using 35% polyethyleneglycol and cultured in 96-well culture plates. Hybridomas resulting fromthe fusion were selected in HAT medium. Ten days after the fusion,hybridoma culture supernatants were screened in an ELISA to test for thepresence of monoclonal antibodies binding to the Apo-2 ECD immunoadhesinprotein.

[0303] In the ELISA, 96-well microtiter plates (Maxisorb; Nunc,Kamstrup, Denmark) were coated by adding 50 μl of 2 μg/ml goatanti-human IgG Fc (purchased from Cappel Laboratories) in PBS to eachwell and incubating at 4° C. overnight. The plates were then washedthree times with wash buffer (PBS containing 0.05% Tween 20). The wellsin the microtiter plates were then blocked with 50 μl of 2.0% bovineserum albumin in PBS and incubated at room temperature for 1 hour. Theplates were then washed again three times with wash buffer.

[0304] After the washing step, 50 μl of 0.4 μg/ml Apo-2 ECDimmunoadhesin protein (as described above) in assay buffer was added toeach well. The plates were incubated for 1 hour at room temperature on ashaker apparatus, followed by washing three times with wash buffer.

[0305] Following the wash steps, 100 μl of the hybridoma supernatants orpurified antibody (using Protein A-sepharose columns) (1 μg/ml) wasadded to designated wells in the presence of CD4-IgG. 100 μl ofP3X63AgU.1 myeloma cell conditioned medium was added to other designatedwells as controls. The plates were incubated at room temperature for 1hour on a shaker apparatus and then washed three times with wash buffer.

[0306] Next, 50 μl HRP-conjugated goat anti-mouse IgG Fc (purchased fromCappel Laboratories), diluted 1:1000 in assay buffer (0.5% bovine serumalbumin, 0.05% Tween-20, 0.01% Thimersol in PBS), was added to each welland the plates incubated for 1 hour at room temperature on a shakerapparatus. The plates were washed three times with wash buffer, followedby addition of 50 μl of substrate (TMB microwell peroxidase substrate,Kirkegaard & Perry, Gaithersburg, Md.) to each well and incubation atroom temperature for 10 minutes. The reaction was stopped by adding 50μl of TMB 1-component stop solution (diethyl glycol, Kirkegaard & Perry)to each well, and absorbance at 450 nm was read in an automatedmicrotiter plate reader.

[0307] Of the hybridoma supernatants screened in the ELTSA, 22supernatants tested positive (calculated as approximately 4 times abovebackground). The supernatants testing positive in the ELISA were furtheranalyzed by FACS analysis using 9D cells (a human B lymphoid cell lineexpressing Apo-2; Genentech, Inc.) and FITC-conjugated goat anti-mouseIgG. For this analysis, 25 μl of cells suspended (at 4×10⁶ cells/ml) incell sorter buffer (PBS containing 1% FCS and 0.02% NaN₃) were added toU-bottom microtiter wells, mixed with 100 μl of culture supernatant orpurified antibody (purified on Protein A-sepharose columns) (10 μg/ml)in cell sorter buffer, and incubated for 30 minutes on ice. The cellswere then washed and incubated with 100 μl FITC-conjugated goatanti-mouse IgG for 30 minutes at 4° C. Cells were then washed twice,resuspended in 150 μl of cell sorter buffer and then analyzed by FACScan(Becton Dickinson, Mountain View, Calif.). FACS analysis showed 8/22supernatants were positive for anti-Apo-2 antibodies.

[0308]FIG. 7 shows the FACS staining of 9D cells incubated with one ofthe Apo-2 antibodies, referred to as 3F11.39.7. As shown in FIG. 7, the3F11.39.7 antibody recognizes the Apo-2 receptor expressed in 9D cells.

Example 10

[0309] Assay for Ability of Apo-2 Abs to Agonistically Induce Apoptosis

[0310] Hybridoma supernatants and purified antibodies (as described inExample 9 above) were tested for activity to induce Apo-2 mediated 9Dcell apoptosis. The 9D cells (5×10⁵ cells/0.1ml) were incubated withvarying concentrations of antibodies in 100 μl complete RPMI media at 4°C. for 15 minutes. The cells were then incubated for 5 minutes at 37° C.and 10 μg of goat anti-mouse IgG Fc antibody (Cappel Laboratories) in300 μl of complete RPMI was added to some of the cell samples. At thispoint, the cells were incubated overnight at 37° C. and in the presenceof 7% CO₂ The cells were then harvested and washed once with PBS. Theviability of the cells was determined by staining of FITC-annexin Vbinding to phosphatidylserine according to manufacturer recommendations(Clontech). The cells were washed in PBS and resuspended in 200 μlbinding buffer. Ten μl of annexin-V-FITC (1 μg/ml) and 10 μl ofpropidium iodide were added to the cells. After incubation for 15minutes in the dark, the 9D cells were analyzed by FACS.

[0311] As shown in FIG. 8, the 3F11.39.7 antibody (in the absence of thegoat anti-mouse TgG Fc) induced apoptosis in the 9D cells as compared tothe control antibodies. Agonistic activity, however, was enhanced byApo-2 receptor cross-linking in the presence of the goat anti-mouse IgGFc (see FIG. 9). This enhanced apoptosis (FIG. 9) by the combination ofantibodies is comparable to the apoptotic activity of Apo-2L in 9D cells(data not shown).

Example 11

[0312] Assay for Antibody Ability to Block Apo-2 Ligand-InducedApoptosis

[0313] Hybridoma supernatants and purified antibodies (as described inExample 9 above) were tested for activity to block Apo-2 ligand induced9D cell apoptosis. The 9D cells (5×10⁵ cells/0.1 ml) were suspended incomplete RPMI media (RPMI plus 10%FCS, glutamine, nonessential aminoacids, penicillin, streptomycin, sodium pyruvate) and placed intoindividual Falcon 2052 tubes. Cells were then incubated with 10 μg ofantibodies in 200 μl media for 15 minutes on ice. 0.2 ml of Apo-2 ligand(2.5 μg/ml) (soluble His-tagged Apo-2L prepared as described in WO97/25428; see also Pitti et al., supra) was suspended into complete RPMImedia, and then added into the tubes containing the 9D cells. The 9Dcells were incubated overnight at 37° C. and in the presence of 7% CO₂The incubated cells were then harvested and washed once with PBS. Theviability of the cells was determined by staining of FITC-annexin Vbinding to phosphatidylserine according to manufacturer recommendations(Clontech). Specifically, the cells were washed in PBS and resuspendedin 200 μl binding buffer. Ten μl of annexin-V-FITC (1 μg/ml) and 10 μlof propidium iodide were added to the cells. After incubation for 15minutes in the dark, the 9D cells were analyzed by FACS.

[0314] The results are shown in FIG. 10. Since 9D cells 2b express morethan one receptor for Apo-2L, Apo-2L can induce apoptosis in the 9Dcells by interacting with either Apo-2 or the DR4 receptor. Thus, todetect any blocking activity of the Apo-2 antibodies, the interactionbetween DR4 and Apo-2L needed to be blocked. In combination with theanti-DR4 antibody, 4H6.17.8 (ATCC HB-12455), the Apo-2 antibody3F11.39.7 was able to block approximately 50% of apoptosis induced byApo-2L. The remaining approximately 50% apoptotic activity is believedto be due to the agonistic activities of these two antibodies bythemselves, as shown in FIG. 10. Accordingly, it is believed that the3F11.39.7 antibody is a blocking Apo-2 antibody or an antibody whichbinds Apo-2 in a mode which competes with binding of Apo-2 ligand toApo-2.

Example 12

[0315] 35 ELISA Assay to Test Binding of Apo-2 Antibodies to Other Apo-2Ligand Receptors

[0316] An ELISA was conducted to determine if the monoclonal antibodydescribed in Example 9 was able to bind other known Apo-2L receptorsbeside Apo-2. Specifically, the 3F11.39.7 antibody was tested forbinding to DR4 [Pan et al., supra], DcR1 [Sheridan et al., supra], andDcR2 [Marsters et al., Curr. Biol., 7:1003-1006 (1997)]. The ELISA wasperformed essentially as described in Example 9 above.

[0317] The results are shown in FIG. 11. The Apo-2 antibody 3F11.39.7bound to Apo-2. The 3F11.39.7 antibody also showed some cross-reactivityto DR4, but not to DcR1 or DcR2.

Example 13

[0318] Antibody Isotyping

[0319] The isotype of the 3F11.39.7 antibody (as described above) wasdetermined by coating microtiter plates with isotype specific goatanti-mouse Ig (Fisher Biotech, Pittsburgh, Pa.) overnight at 4° C. Theplates were then washed with wash buffer (as described in Example 9above). The wells in the microtiter plates were then blocked with 200 μlof 2% bovine serum albumin (BSA) and incubated at room temperature forone hour. The plates were washed again three times with wash buffer.Next, 100 μl of 5 μg/ml of purified 3F11.39.7 antibody was added todesignated wells. The plates were incubated at room temperature for 30minutes and then 50 μl HRP-conjugated goat anti-mouse IgG (as describedabove) was added to each well. The plates were incubated for 30 minutesat room temperature. The level of HRP bound to the plate was detectedusing HRP substrate as described above.

[0320] The isotyping analysis showed that the 3F11.39.7 antibody is anIgGl antibody.

Example 14 Single-Chain Apo-2 Antibodies

[0321] A. Antibody Phage Selection using Streptavidin-CoatedParamagnetic Beads

[0322] A phage library was selected using soluble biotinylated antigenand streptavidin-coated paramagnetic beads. The antigen, an Apo-2 ECDimmunoadhesin prepared as described in Example 2B above, wasbiotinylated using IMMUNOPURE NHS-biotin (biotiny-N-hydroxy-succinimide,Pierce) according to manufacturer's instructions.

[0323] Two panning experiments were performed. The first experiment wasdesigned to isolate phage clones specific for Apo-2 and which did notcross react with DR4 or DcR1. Three rounds of panning were carried out.For the first round, 10 μl of the Cambridge Antibody Technologies phagelibrary were blocked with 1 ml of MPBST (3% dry milk powder, 1×PBS, 0.2%TWEEN) containing 800 μg of CD4-Ig, 300 μg DR4-Ig, and 200 μg of DcR1-Igfor 1 hour on a rotating wheel at room temperature (CD4-Ig, DR4, andDcR1 are described in Capon et al., Nature, 337:525 (1989); Pan et al.,supra; and Sheridan et al., supra). Biotinylated Apo-2 ECD immunoadhesinwas then added to a final concentration of 100 nM, and phage wereallowed to bind antigen for 1 hour at 37° C. Meanwhile, 300 μl ofDYNABEADS M-280, coated with streptavidin (DYNAL) were washed 3 timeswith 1 ml MPBST (using a DYNAL Magnetic Particle Concentrator) and thenblocked for 2 hours at 37° C. with 1 ml fresh MPBST on a rotator. Thebeads were collected with the MPC, resuspended in 50 μl of MPBST, andadded to the phage-plus-antigen solution. Mixing continued on a wheel atroom temperature for 15 minutes. The DYNABEADS and attached phage werethen washed a total of 7 times: 3 times with 1 ml PBS-TWEEN, once withMPBS, followed by 3 times with PBS.

[0324] Phage were eluted from the beads by incubating 5 minutes at roomtemperature with 300 μl of 100 mM triethylamine. The phage-containingsupernatant was removed and neutralized with 150 μl of 1 M Tris-HCl (pH7.4). Neutralized phage were used to infect mid-log TG1 host cells andplated on 2YT agar supplemented with 2% glucose and 100 μg/mlcarbenicillin. After overnight growth at 30° C., colonies were scrapedinto 10 ml 2YT. 50 μl of this solution was used to inoculate 25 ml of2YT with carbenicillin and glucose and incubated, shaking, for 2 hoursat 37° C. Helper phage M13KO7 (Pharmacia) were added at a m.o.i. of 10.After adsorption, the cells were pelleted and resuspended in 25 ml of2YT with carbenicillin (100 μg/ml) and kanamycin (50 μg/ml) and growthcontinued at 30° C. for 4 hours. E. coli were removed from the phage bycentrifugation, and 1 ml of these phage (approximately 10¹² c.f.u.) wereused in subsequent rounds of selection.

[0325] For the second round of selection, the 1 ml of harvested phagewas adjusted to 3% dry milk, IX PBS, 0.2% TWEEN and then 100 μg DR4-Ig,65 μg DcR1-Ig, and 500 μg of CD4-Ig were added for blocking. Forselection, biotinylated Apo-2 was added at 10 nM. Washing stringency wasincreased to two cycles of 7 washes.

[0326] For the third round of selection, phage were blocked with onlyMPBST. Biotinylated Apo-2 was added to 1 nM, and washing stringency wasincreased to three cycles of 7 washes. Relatively few clones wereobtained in this round; therefore Pan 2B, Round 3 was performed using 5nM of biotinylated Apo-2 with all other conditions repeated as before.

[0327] A second panning experiment was performed similarly as aboveexcept that in Rounds 1 and 2, blocking of phage solutions was conductedwith MPBST containing 1.0 mg/ml CD4-Ig (no other immunoadhesins) andRound 3 was blocked with MPBST only. Biotinylated Apo-2 was added at 200nM in Round 1, 60 nM in Round 2, and 12 nM in Round 3. At each round,phage were eluted from the magnetic beads with 300 μl of 100 nMtriethylamine, then with 300 μl 0.1 M Tris-HCl (pH 7.5), and then with300 μl glycine-0.1 M HCl (pH 2.2) containing 1 mg/ml BSA. The phageobtained from the three sequential elutions were pooled and used toinfect host strain TG1 as above.

[0328] B. ELISA Screening of Selected Clones

[0329] After each round of selection, individual carbenicillin-resistantcolonies were screened by ELISA to identify those producingApo-2-binding phage. Only those clones which were positive in two ormore assay formats were further studied.

[0330] Individual clones were inoculated into 2TY with 2% glucose and100 μg/ml carbenicillin in 96-well tissue culture plates and grown untilturbid. Cultures were then infected at a m.o.i. of 10 with M12KO7 helperphage, and infected cells were transferred to 2YT media containingcarbenicillin (100 μg/ml) and kanamycin (50 μg/ml) for growth overnightat 30° C. with gentle shaking.

[0331] NUNC MAXISORP microtiter plates were coated with 50 μl per wellof Apo-2 ECD immunoadhesin, or CD4-IgG, at 2 μg/ml in 50 mM carbonatebuffer (pH 9.6), at 4° C. overnight. After removing antigen, plates wereblocked with 3% dry milk in PBS (MPBS) for 2 hours at room temperature.

[0332] Phage cultures were centrifuged and 100 μl of phage-containingsupernatants were blocked with 20 μl of 6×PBS/18% dry milk for 1 hour atroom temperature. Block was removed from titer plates and blocked phageadded and allowed to bind for 1 hour at room temperature. After washing,phage were detected with a 1:5000 dilution of horseradishperoxidase-conjugated anti-M13 antibody (Pharmacia) in MPBS followed by3′,3′,5′,5′-tetramethylbenzidine (TMB). Reactions were stopped by theaddition of H₂SO₄ and readings taken by subtracting the A_(405 nm) fromthe A_(450 nm).

[0333] C. DNA Fingerprinting of Clones

[0334] The diversity of Apo-2-binding clones was determined by PCRamplifying the scFv insert using primers pUC19R (5′AGC GGA TAA CAA TTTCAC ACA GG 3′) (SEQ. ID. NO:12) which anneals upstream of the leadersequence and fdtetseq (5′GTC GTC TTT CCA GAC GGT AGT 3′) (SEQ. ID.NO:13) which anneals in the 5′ end of gene III, followed by digestionwith the frequent-cutting restriction enzyme BstNI.

[0335] DNA Fingerprinting: Protocol Mix A: dH2O 67 μl 10 x ampliTaqbuffer 10 25 mM MgCl₂ 10 DMSO, 50% 2 forward primer 1 Mix B: 2.5 mMdNTPs 8 μl AMPLITAQ 0.5 reverse primer 1.0

[0336] 90 μl of Mix A was placed in a reaction tube and then inoculatedwith a very small portion of E. coli colony using a yellow tip. Thereaction mix was then heated in a PCR block to 98° C., for 3 minutes,removed, and placed on ice. 10 ul Mix B was then added and the reactionmix was thermocycled at 95° C., 30 sec, 55° C. 30 sec, 72° C. 1 minute20 sec, for 25 cycles in a Perkin Elmer 2400 thermocycler. 10 μl of theresultant reaction product was then removed and run on a 1% agarose gelto test for a 1 kB band. The remaining mix was brought to 1×BstNIreaction buffer, 5 units BstNI was added and the DNA was allowed todigest for 2 hours at 60° C. The resultant samples were thenelectrophoresed on a GeneGel Excel 12.5% acrylamide gel (PharmaciaBiotech).

[0337] D. Sequencing of Clones

[0338] The nucleotide sequence of representative clones of eachfingerprint pattern were obtained. Colonies were inoculated into 50 mlof LB medium supplemented with 2% glucose and 100 μg/ml carbenicillin,and grown overnight at 30° C. DNA was isolated using Qiagen Tip-100s andthe manufacturer's protocol and cycle sequenced with fluorescent dideoxychain terminators (Applied Biosystems). Samples were run on an AppliedBiosystems 373A Automated DNA Sequencer and sequences analyzed using theprogram “Sequencher” (Gene Codes Corporation). The nucleotides sequencesof selected antibodies 16E2, 20E6 and 24C4 are shown in SEQ ID NO:6, SEQID NO:7, and SEQ ID NO:8, respectively, (in FIGS. 15A, 15B and 15Crespectively). The corresponding amino acid sequences of antibodies16E2, 20E6 and 24C4 are shown in SEQ ID NO:9, SEQ ID NO:10, and SEQ IDNO:11, respectively (and in FIG. 16). In addition, FIG. 16 identifiesthe signal region, and heavy and light chain complementarity determiningregions (underlined) of these scFv molecules. The CDR regions shown inFIG. 16 were assigned according to the methods of Kabat et al.,“Sequences of Proteins of Immunological Interest,” NIH Publ. No.91-3242, 5^(th) Edition.

[0339] E. Purification of scFvs with (his)₆

[0340] For protein purification of soluble antibody, E. coli strain 33D3was transformed with phagemid DNA. Five ml of 2YT with carbenicillin andglucose was used to grow overnight cultures at 30° C. 2.5 ml of thesecultures were diluted into 250 ml of the same media and grown to anOD₆₀₀ of approximately 1.2. The cells were pelleted and resuspended in500 ml of 2YT containing IPTG (1 mM) and carbenicillin (100 μg/ml) toinduce expression and grown for a further 16 hours at 22° C. Cellpellets were harvested and frozen at −20° C.

[0341] The antibodies were purified by immobilized metal chelateaffinity chromatography (IMAC). Frozen pellets were resuspended in 10 mlof ice-cold shockate buffer (25 mM TRIS-HCl, 1 mM EDTA, 500 mM NaCl, 20%sucrose, 1 mM PMSF) by shaking on ice for 1 hour. Imidazole was added to20 mM, and cell debris removed by centrifugation. The supernatants wereadjusted to 1 mM MgCl₂ and 50 mM phosphate buffer pH 7.5. Ni-NTA agaroseresin from Qiagen was used according to the manufacturer's instructions.The resin was equilibrated with 50 mM sodium phosphate buffer pH 7.5,500 mM NaCl, 20 mM imidazole, and the shockate added. Binding occurredin either a batch mode or on a gravity flow column. The resin was thenwashed twice with 10 bed volumes of equilibration buffer, and twice withbuffer containing imidazole increased to 50 mM. Elution of proteins waswith 50 mM phosphate buffer pH 7.5, 500 mM NaCl and 250 mM imidazole.Excess salt and imidazole was removed on a PD-10 column (Pharmacia), andproteins were concentrated using a Centricom 10 to a volume of about 1ml.

[0342] Concentration was estimated spectrophotometrically assuming anA280 nm of 1.0=0.6 mg/ml.

[0343] F. Assays to Determine Binding Specificity of Anti-Apo-2 scFvs

[0344] To evaluate the specificity of each of the scfv clones, ELISAassays were performed to evaluate binding of 16E2, 20E6 and 24C4 toApo-2 ECD-Ig, DR4-Ig, DcR1-Ig, DcR2-Ig and CD4-Ig (described above andin Example 12).

[0345] In brief, NUNC ELISA plates were coated with 50 μl of a 1 μg/mlreceptor-Ig immunoadhesin molecule in 0.05 M sodium carbonate buffer, pH9.5, and allowed to incubate overnight at 4° C. Plates were then blockedwith 285 μl ELISA diluent (PBS supplemented with 0.5% BSA, 0.05% Tween20, pH 7.4) for at least one hour at room temperature. 50 μl of thescFvs were added to the plates in a 1:5 serial dilution and allowed toincubate for 1 hour at room temperature. After this 1 hour dilution, theplates were washed 6 times with PBS/0.05% Tween. After binding toantigen coated plates, soluble scFv was detected by adding 50 pl of 1μg/ml Mab 9E10 (an anti-c-myc antibody; ATCC CRL 1729) per well andallowing the plates to incubate for 1 hour at room temperature. Afterwashing the plates 6 times with PBS/0.05% Tween, 50 μl of a 1:5000dilution of horseradish peroxidase-conjugated anti-Murine IgG antibody(Cappel catalogue: 55569) in MPBS was added to the plates and allowed toincubate for 1 hour. An observable signal was generated by adding 50 μlof 3′,3′,5′,5′-tetramethylbenzidine (TMB) peroxidase substrate (KPLcatalogue #: 50-76-00). Reactions were stopped by the addition of H₂SO₄and readings taken by subtracting the A_(405 nm) from the A_(450 nm).

[0346] As illustrated in FIGS. 12A, 12B and 12C, the ELISA assays showedthat each of these antibodies exhibited a relatively high degree ofspecificity for Apo-2.

[0347] Additional assays utilizing transfected cells also showed thespecificity of 16E2 antibody for Apo-2. Specifically,immunohistochemistry experiments were performed to evaluate the bindingspecificity of the 16E2 antibody to Apo-2 and DR4-transfected CHO cells.CHO cells were transfected with vector alone or vector containing thegene for Apo-2 or DR4. The transfected cells were removed from cultureplates, pelleted, and washed twice with PBS. The pellets were thenresuspended in O.C.T. (Fisher), flash frozen in isopentain and LN₂, andlater sectioned using standard protocols. Staining of the sectionedcells was performed using a Vectastain Elite ABC kit. The sections wereincubated with either anti-Apo-2 antibody 16E2 or a negative controlsingle chain antibody. The secondary antibody employed was either abiotinylated anti-c-myc 9E10 antibody or anti-Penta His antibody(Qiagen) followed by biotinylated anti-mouse IgG.

[0348] This immunohistochemistry assay showed specific staining of theApo-2-transfected cells but not the DR4-transfected cells. The cellularstaining was predominantly cytoplasmic.

Example 15

[0349] Assay for Ability of His-Tagged scFvs to Agonistically InduceApoptosis

[0350] A. Annexin V-biotin/Streptavidin-[S-35] 96 Well Assays

[0351] Purified scFv antibodies (as described in Example 14 above) weretested for ability to induce Apo-2 mediated apoptosis.

[0352] In brief, SK-MES-1 cells (human lung carcinoma cell line; ATCCHTB 58) or HCT 116 cells (human colon carcinoma cell line; ATCC CCL 247)(4×10⁴ cells/well) were aliquoted into 96 well plates in assay medium(1:1 mixture of phenol-red free Dulbecco modified Eagle medium andphenol-red free Ham's F-12 nutrient mixture supplemented with 10% fetalbovine serum, 2 mM L-glutamine, 100 U/ml penicillin and 100 ug/mlstreptomycin) and allowed to attach overnight at 37° C. The media wasthen removed and 0.1 ml of assay medium containing scFv at a finalconcentration of 50 ug/ml (16E2 or 20E6) was added to the wells (serialdilutions of 1:2 performed in the plates) and allowed to incubate for 1hour at room temperature. Other single chain antibodies were used asnegative controls: an anti-tissue factor scFv clone, 7D5, or a scFvreferred to as 19B8. After the 1 hour incubation with scFv antibody, 0.1ml of 10 ug/ml anti-His (Qiagen, cat. No. 1007671) or anti-c-mycantibodies were added to the appropriate wells. Wells not receiving acrosslinking antibody received media alone. The plates were then allowedto incubate for 30 minutes at room temperature. After the 30 minutesincubation, 0.1 ml of 10 ug/ml goat anti-mouse IgG (ICN cst. No. 67-029)was added to the appropriate wells. Wells not receiving anti-IgGantibody received media alone. The plates were then placed in anincubator for 15 minutes to allow the pH to return to 7.0. For positivecontrols, a 2 ug/ml solution of Apo-2 ligand (Apo-2L) (prepared asdescribed in Example 11) in potassium phosphate buffer at pH 7.0 wasadded to the appropriate wells, with serial 2 fold dilutions carried outin the plate. The negative control wells received media alone. The cellswere then incubated overnight at 37° C. in the presence of 5% CO₂. 0.05ml of annexin V-biotin (1 ug/ml) in 2×Ca²⁺ binding buffer (NeXins B.V.)was then added to the wells and then allowed to mix on a shaker for 30minutes. 0.05 ml of strepavidin-[S-35] (final concentration of 2.5×10⁴cpm/well) (Amersham) in 2×Ca²⁺ binding buffer was then added to thewells and then allowed to mix on a shaker for 30 minutes. The plateswere then sealed and centrifuged for 4 minutes at 1500 rpm. To assessthe extent of apoptosis, the plates were then counted on a TrialuxMicrobeta Counter (Wallace) to obtain cpm values corresponding toAnnexin-V binding.

[0353] As shown in FIGS. 13C and 14B, the 16E2 and 20E6 antibodiesagonistically induced apoptosis in SK-MES-1 cells.

[0354] B. Crystal Violet Assays

[0355] In addition to the annexin V-biotin/streptavidin-[S-35] assaydescribed above, scFv antibodies (as described in Example 14 above) weretested for activity to induce Apo-2 mediated apoptosis via assaysutilizing crystal violet.

[0356] In brief, the SK-MES-1 cells were plated at 4×10⁴ cells/well inassay medium (described in Section A above) and allowed to attachovernight at 37° C. The medium was removed and 0.1 ml of assay mediumcontaining scFv (as described in Section A above) at a finalconcentration of 50 μg/ml was added to the appropriate wells (wellswithout scFv added receive a media change). Selected wells received“pre-complexed” samples in which 10 ug/ml scFv 16E2 was combined with100 ug/ml anti-His antibody for 5 hours at 4° C. with continuous mixingbefore addition to the plate. The plates were allowed to incubate for 1hour at room temperature.

[0357] The scFv medium was removed and 0.1 ml of 10 μg/ml anti-His(Qiagen, cat. no. 1007671) or anti-c-myc antibodies diluted in assaymedium was added to the wells (wells without crosslinker receive a mediachange.) The plates were then allowed to incubate for 30 minutes at roomtemperature.

[0358] The medium was then removed and 0. 1 ml of 10 μg/ml Goatanti-Mouse IgG (Fc Fragment specific-ICN cst. no. 67-029) diluted inassay medium was added to the appropriate wells (wells without anti-Fcreceive a media change). The plates were then placed in the incubatorfor 15 minutes to allow the pH to return to 7.0.

[0359] Apo-2L (stock at 100 μg/ml in potassium phosphate buffer pH 7.0)was diluted to 2 μg/ml and 0.1 ml was added to the appropriate wells.Serial two-fold dilutions were carried down the plate. The plates werethen incubated overnight at 37° C.

[0360] All medium was removed from the wells and the plates were thenflooded with crystal violet solution. The plates were allowed to stainfor 15 minutes. The crystal violet was removed by flooding the plateswith running tap water. The plates were then allowed to dry overnight.

[0361] The plates were read on an SLT plate reader at 540 nm and thedata analyzed using an Excel macro and 4p-fit.

[0362] As shown in FIGS. 13A, 13B, 14A and 14B, the 16E2 and 20E6antibodies agonistically induced apoptosis in SK-MES-1 cells.

Example 16

[0363] Assay for Ability of gD-Tagged scFvs to Agonistically InduceApoptosis

[0364] A purified gD-tagged form of 16E2 scFv was tested for ability toinduce Apo-2 mediated apoptosis in a crystal violet assay as describedin Example 15 above.

[0365] A. Construction of scFv with gD Tag

[0366] The Sfi I to Not I fragment of the scFv form of 16E2 wassubcloned into a derivative of pAK19 (Carter et al., Methods:A Companionto Methods in Enzymology, 3:183-192 (1991)] containing the phoA promoterand stII signal sequence rather than the lacz promoter and hybrid signalsequence of the original library. For ease of purification, a DNAfragment coding for 12 amino acids(met-ala-asp-pro-asn-arg-phe-arg-gly-lys-asp-leu SEQ ID NO:14) derivedfrom herpes simplex virus type 1 glycoprotein D (Lasky et al., DNA,3:23-29 (1984)) was synthesized and inserted at the 3′ end of the VLdomain in place of the (his)₆ and c-myc epitope originally present inthe Cambridge Antibody Technologies library clones.

[0367] B. Expression in E. coli

[0368] The plasmid containing the gene for scFv 16E2-gD was transformedinto E. coli strain 33D3 for expression in shake flask cultures. 5 ml of2YT with carbenicillin and glucose was used to grow overnight culturesat 30° C. 2.5 ml of these cultures were diluted into 250 ml of the samemedium and grown to an OD₆₀₀ of approximately 1.0. The cells werepelleted and resuspended in 500 ml of Modified AP-5 Minimal Mediacontaining carbenicillin (100 μg/ml) and grown for an additional 16hours at 30° C. The cells were then pelleted and frozen.

[0369] C. Purification of scFv with gD Tag

[0370] Frozen cell paste was resuspended at 1 gm/10 ml of shockatebuffer (25 mM Tris-HCl, 1 mM EDTA, 500 mM NaCl, 20% sucrose, 1 mM PMSF,pH 7.2) and gently agitated 4 hours on ice. The cell suspension was thenprocessed through a Polytron microfluidizer (Brinkman). Cell debris wasremoved by centrifugation at 10,000×g for 30 minutes. After filtrationthrough a 0.22 micron filter, the supernatant was loaded onto anaffinity column (2.5×9.0 cm) consisting of an anti-gD antibody 5B6(Paborsky et al., Protein Engineering, 3:547-553 (1990)) coupled to CNBrSepharose which had been equilibrated with PBS. The column was washed 18hours with PBS until the absorbance of the column effluent wasequivalent to baseline. All steps were done at 4° C. at a linear flowrate of 25 cm/hour. Elution was performed with 0.1 M acetic acid, 0.5 MNaCl, pH 2.9. Column fractions were monitored by absorbance at 280 nmand peak fractions pooled, neutralized with 1.0 M Tris, pH 8.0, dialyzedagainst PBS and sterile filtered. The resultant protein preparationswere analyzed by non-reducing SDS-PAGE.

[0371] D. Crystal Violet Assay

[0372] The apoptosis assay was performed essentially as described inExample 15(B) above except that samples were serially diluted 1:3 in theplates and the 16E2-gD tagged antibody was tested in addition to twoother preparations of 16E2 scFv (referred to as Prep. A and Prep. B inFIG. 14C). The results of the assay showing apoptosis induction inSK-MES-1 cells by 16E2-gD antibody are illustrated in FIG. 14C.

[0373] Deposit of Material

[0374] The following materials have been deposited with the AmericanType Culture Collection, 10801 University Boulevard, Manassas, Va., USA(ATCC): Material ATCC Dep. No. Deposit Date pRK5-Apo-2 209021 May 8,1997 3F11.39.7 HB-12456 Jan. 13, 1998

[0375] This deposit was made under the provisions of the Budapest Treatyon the International Recognition of the Deposit of Microorganisms forthe Purpose of Patent Procedure and the Regulations thereunder (BudapestTreaty). This assures maintenance of a viable culture of the deposit for30 years from the date of deposit. The deposit will be made available byATCC under the terms of the Budapest Treaty, and subject to an agreementbetween Genentech, Inc. and ATCC, which assures permanent andunrestricted availability of the progeny of the culture of the depositto the public upon issuance of the pertinent U.S. patent or upon layingopen to the public of any U.S. or foreign patent application, whichevercomes first, and assures availability of the progeny to one determinedby the U.S. Commissioner of Patents and Trademarks to be entitledthereto according to 35 USC Section 122 and the Commissioner's rulespursuant thereto (including 37 CFR Section 1.14 with particularreference to 886 OG 638).

[0376] The assignee of the present application has agreed that if aculture of the materials on deposit should die or be lost or destroyedwhen cultivated under suitable conditions, the materials will bepromptly replaced on notification with another of the same. Availabilityof the deposited material is not to be construed as a license topractice the invention in contravention of the rights granted under theauthority of any government in accordance with its patent laws.

[0377] The foregoing written specification is considered to besufficient to enable one skilled in the art to practice the invention.The present invention is not to be limited in scope by the constructdeposited, since the deposited embodiment is intended as a singleillustration of certain aspects of the invention and any constructs thatare functionally equivalent are within the scope of this invention. Thedeposit of material herein does not constitute an admission that thewritten description herein contained is inadequate to enable thepractice of any aspect of the invention, including the best modethereof, nor is it to be construed as limiting the scope of the claimsto the specific illustrations that it represents. Indeed, variousmodifications of the invention in addition to those shown and describedherein will become apparent to those skilled in the art from theforegoing description and fall within the scope of the appended claims.

What is claimed is:
 1. Isolated Apo-2 polypeptide having at least 80%amino acid sequence identity with native sequence Apo-2 polypeptidecomprising amino acid residues 1 to 411 of SEQ ID NO:1.
 2. The Apo-2polypeptide of claim 1 wherein said polypeptide has at least 90% aminoacid sequence identity.
 3. The Apo-2 polypeptide of claim 2 wherein saidpolypeptide has at least 95% amino acid sequence identity.
 4. IsolatedApo-2 polypeptide comprising amino acid residues 1 to 411 of SEQ IDNO:1.
 5. Isolated extracellular domain sequence of Apo-2 polypeptidecomprising amino acid residues 54 to 182 of SEQ ID NO:1.
 6. Theextracellular domain sequence of claim 5 comprising amino acid residues1 to 182 of SEQ ID NO:1.
 7. Isolated death domain sequence of Apo-2polypeptide comprising amino acid residues 324 to 391 of SEQ ID NO:1. 8.A chimeric molecule comprising the Apo-2 polypeptide of claim 1 or theextracellular domain sequence of claim 5 fused to a heterologous aminoacid sequence.
 9. The chimeric molecule of claim 8 wherein saidheterologous amino acid sequence is an epitope tag sequence.
 10. Thechimeric molecule of claim 8 wherein said heterologous amino acidsequence is an immunoglobulin sequence.
 11. The chimeric molecule ofclaim 10 wherein said immunoglobulin sequence is an IgG.
 12. Isolatednucleic acid comprising a DNA encoding the polypeptide of claim 1, theextracellular domain sequence of claim 5, or the death domain sequenceof claim
 7. 13. The nucleic acid of claim 12, wherein said DNA encodesan Apo-2 polypeptide comprising amino acid residues 1 to 411 of SEQ IDNO:1.
 14. A vector comprising the nucleic acid of claim
 12. 15. Thevector of claim 14 operably linked to control sequences recognized by ahost cell transformed with the vector.
 16. The vector of claim 14comprising ATCC deposit accession number
 209021. 17. A host cellcomprising the vector of claim
 14. 18. The host cell of claim 17comprising a CHO cell.
 19. The host cell of claim 17 comprising E. coli.20. The host cell of claim 17 comprising a yeast cell.
 21. A process ofproducing an Apo-2 polypeptide comprising culturing the host cell ofclaim 17 under conditions sufficient to express Apo-2 polypeptide andrecovering the expressed Apo-2 polypeptide from the culture.
 22. AnApo-2 polypeptide which is obtained or obtainable by expressing thepolypeptide encoded by the cDNA insert in ATCC deposit accession number209021.
 23. A non-human, transgenic animal which contains cells thatexpress DNA encoding Apo-2 polypeptide.
 24. The animal of claim 23 whichis a mouse or rat.
 25. A non-human, knockout animal which contains cellshaving an altered gene encoding Apo-2 polypeptide.
 26. The animal ofclaim 25 which is a mouse or rat.
 27. An antibody which specificallybinds to an Apo-2 polypeptide.
 28. The antibody of claim 27 which is amonoclonal antibody.
 29. The antibody of claim 27 comprising anagonistic antibody.
 30. The antibody of claim 27 comprising a blockingantibody.
 31. The antibody of claim 24 comprising a chimeric antibody.32. The antibody of claim 28 wherein said antibody is an IgG antibody.33. The antibody of claim 28 wherein said antibody comprises an Fabfragment.
 34. The antibody of claim 28 wherein said antibody comprises ascFv fragment.
 35. The antibody of claim 28 wherein said antibodycomprises a F(ab′)2 fragment.
 36. The antibody of claim 27 wherein saidantibody comprises a human antibody.
 37. The antibody of claim 28 havingthe biological characteristics of the monoclonal antibody produced bythe hybridoma cell line deposited as ATCC accession number HB-12456. 38.The antibody of claim 28 wherein the antibody binds to the same epitopeas the epitope to which the monoclonal antibody produced by thehybridoma cell line deposited as ATCC accession number HB-12456 binds.39. A hybridoma cell line which produces the antibody of claim
 28. 40.The hybridoma cell line deposited as ATCC accession number HB-12456. 41.The monoclonal antibody produced by the hybridoma cell line deposited asATCC accession number HB-12456.
 42. The antibody of claim 27 whereinsaid antibody is a single-chain antibody.
 43. The antibody of claim 42wherein said antibody comprises the 16E2 antibody.
 44. The antibody ofclaim 42 wherein said antibody comprises the 20E6 antibody.
 45. Theantibody of claim 42 wherein said antibody comprises the 24C4 antibody.46. The antibody of claim 42 wherein said antibody is fused to anepitope tag sequence.
 47. A chimeric molecule comprising the antibody ofclaim 27 fused to a heterologous amino acid sequence.
 48. The chimericmolecule of claim 47 wherein said heterologous amino acid sequencecomprises an immunoglobulin sequence.
 49. A dimeric molecule comprisingthe Apo-2 antibody of claim 27 and a heterologous antibody.
 50. Ahomodimeric molecule comprising a first Apo-2 antibody and a secondApo-2 antibody.
 51. Isolated nucleic acid comprising DNA encoding theApo-2 antibody of claim
 43. 52. Isolated nucleic acid comprising DNAencoding the antibody of claim
 44. 53. Isolated nucleic acid comprisingDNA encoding the antibody of claim
 45. 54. A vector comprising thenucleic acid of claim 51, 52, or
 53. 55. A host cell comprising thevector of claim
 54. 56. A method of producing an Apo-2 antibodycomprising culturing the host cell of claim 55 under conditions whereinthe DNA is expressed.
 57. A composition comprising the antibody of claim27 and a carrier.
 58. The composition of claim 57 wherein said carrieris a pharmaceutically-acceptable carrier.
 59. A method of inducingapoptosis in mammalian cancer cells comprising exposing mammalian cancercells to an effective amount of the Apo-2 agonistic antibody of claim29.
 60. The method of claim 59 wherein said agonistic antibody comprisesa single-chain antibody.
 61. A method of treating mammalian cancer cellscomprising exposing mammalian cancer cells to an agent which activatesApo-2.
 62. The method of claim 61 wherein said agent comprises anagonistic Apo-2 antibody.
 63. An article of manufacture comprising acontainer and a composition contained within said container, wherein thecomposition includes Apo-2 polypeptide or Apo-2 antibody.
 64. Thearticle of manufacture of claim 63 further comprising instructions forusing the Apo-2 polypeptide or Apo-2 antibody in vivo or ex vivo.